Methylation Status Of Multiple Genes Research Articles

Methylation Status of Transcriptional Modulatory Genes Associated with Colorectal Cancer in Northeast China.

Background/AimsMethylation status plays a causal role in carcinogenesis in targeted tissues. However, the relationship between the DNA methylation status of multiple genes in blood leukocytes and colorectal cancer (CRC) susceptibility as well as interactions between dietary factors and CRC risks are unclear.MethodsWe performed a case-control study with 466 CRC patients and 507 cancer-free controls to investigate the association among the methylation status of individual genes, multiple CpG site methylation (MCSM), multiple CpG site heterogeneous methylation and CRC susceptibility. Peripheral blood DNA methylation levels were detected by performing methylation-sensitive high-resolution melting.ResultsTotal heterogeneous methylation of CA10 and WT1 conferred a significantly higher risk of CRC (adjusted odds ratio [ORadjusted], 5.445; 95% confidence interval [CI], 3.075 to 9.643; ORadjusted, 1.831; 95% CI, 1.100 to 3.047; respectively). Subjects with high-level MCSM (MCSM-H) status demonstrated a higher risk of CRC (ORadjusted, 4.318; 95% CI, 1.529 to 12.197). Additionally, interactions between the high-level intake of fruit and CRH, WT1, and MCSM on CRC were statistically significant.ConclusionsThe gene methylation status of blood leukocytes may be associated with CRC risk. MCSM-H of blood leukocytes was associated with CRC, especially in younger people. Some dietary factors may affect hypermethylation status and influence susceptibility to CRC.

Incidence and prognostic value of multiple gene promoter methylations in gliomas

Aberrant CpG island methylation is a common phenomenon in malignancy. The methylation status of multiple tumor suppressor genes may serve as a biomarker for early diagnostics and the prediction of prognosis. In this study, we quantitatively determined the promoter methylation status of five tumor-related genes in tumor tissue and paired serum from 240 patients with gliomas. The relationship between hyper-methylation and clinic-pathological parameters was evaluated, and the prognostic value of the methylation status was determined. Hypermethylation in serum was shown to be accompanied by hypermethylation in paired tumor tissues. In both tumors and serum, methylation of polymerase-1 (PARP-1), SHP-1, DAPK-1 and TIMP-3 genes was at significantly higher levels in high-grade compared with low-grade gliomas, indicating that the promoter methylation status positively correlates with tumor grade. In malignant gliomas, the serum methylation levels of PARP-1, and SHP-1 together with IDH-1 mutations were found to be independent prognostic factors for overall survival. Moreover, hypermethylation of PARP-1 in serum correlated with a shorter progression-free survival time. These results suggest that hypermethylation in gliomas correlates with increased malignancy and poor prognosis. Analysis of the serum promoter methylation status of multiple genes could therefore be used as a biomarker for the detection and evaluation of the prognosis of glioma patients.

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

BackgroundQuantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design.MethodsPrimers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings.ResultsUpon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R2=0.985), MAL (R2=0.986) and hsa-miR-124-2 (R2=0.944).ConclusionMultiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions.

Serrated polyposis syndrome and the role of serrated polyps in colorectal cancer development

SUMMARY Serrated polyposis syndrome is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer risk. The mixture of distinct precursor lesion types and malignancies in serrated polyposis syndrome provides a unique model to study the recently proposed serrated neoplasia pathway. This pathway involves the progression of serrated polyps, that is, hyperplastic polyps, sessile serrated adenoma/polyps and/or traditional serrated adenomas, to colorectal cancer. The early genetic events of this route, as currently identified, are BRAF or KRAS mutations and an enhanced CPG island methylation status of multiple genes. There is evidence to suggest that a proportion of sporadic colorectal cancers originate from serrated polyps, which encompass molecular sequences of events such as hypermethylation of different genes and BRAF mutations. This review discusses the characteristics and clinical relevance of serrated polyps and provides an overview of the clinical aspects and treatment of serrated polyposis syndrome.

The application of DNA methylation detection in plasma and tumor tissues in patients with esophageal squamous cell carcinoma

Objective To investigate the methylation status of multiple genes in plasma and tumor tissues and its application in molecular diagnosis of esophageal squamous cell carcinoma (ESCC). Methods Methylation specific polymerase chain reaction (MSP) was used to detect methylation status of 5 tumor suppressor genes，such as adenomatous polyposis coli (APC), retinoic acid receptor-beta2 (RARβ2), E-cadherin (CDH1), cyclin-dependent kinase inhibitor4A (p16INK4α) and ras association domain family member 1A (RASSF1A) in tumour tissues, adjacent normal tissues and plasma which obtained 1 d preoperative and 7 d postoperative in 76 cases with ESCC.60 healthy volunteers were randomly selected as a control which were age-matched and sex-matched.Chi square test was used to analyze DNA methylation rates of 5 genes in various groups of tissue and plasma samples; Kappa test was used to compare the consistency of DNA methylation in the plasma samples and tissue samples, and their correlation was analyzed by Spearman correlation test; Receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity for single gene detection and 5 genes joint detection for diagnosis of esophageal squamous cell carcinoma. Results The methylation rates of APC, RARβ2, CDH1, p16INK4α and RASSF1A in tumour tissues of patients with ESCC were 44.7%（34/76），72.4%（55/76），72.4%（55/76），86.8%（66/76），55.3%（42/76）,respectively, which were significantly higher than that in the corresponding adjacent normal tissues ［6.6%（5/76），3.9%（3/76），3.9%（3/76），3.9%（3/76），2.6%（2/76），χ2=29.01，75.39，75.39，105.34，57.18，all P＜0.001］.The methylation rates of above 5 genes in patients′ plasma were 42.1%（32/76），63.2%（48/76），63.2%（48/76），71.1%（54/76），50.0%（38/76）, respectively, which were significantly higher than that of control group ［3.3%（2/60），3.3%（2/60），1.7%（1/60），3.3%（2/60），1.7%（1/60），χ2=26.88，51.62，55.01，63.48，38.30，all P＜0.001］.The methylation consistency was favorable or well between plasma and tumour tissues in patients with ESCC(Kappa value was 0.679，0.791，0.791，0.542 and 0.895, respectively. all P＜0.001）.In single-gene detection for patients′ plasma，methylation，the sensitivity of 5 genes was 42.1%（32/76），63.2%（48/76），63.2%（48/76），71.1%（54/76），50.0%（38/76）, respectively.The specificity was 96.7%(58/60)，96.7%(58/60)，98.3%(59/60)，96.7%(58/60)，98.3%(59/60), respectively.The area under curve (AUC) of ROC was 0.694 ［95% confidence interval(CI)0.606-0.782）］，0.799（95%CI 0.723-0.875），0.807（95%CI 0.733-0.882），0.839（95%CI 0.769-0.908） and 0.742（95%CI 0.659-0.824）, respectively.In united testing of 5 genes, the sensitivity was 80.3% and the specificity was 88.3%, AUC was 0.843（95%CI 0.773-0.913）.The sensitivity of united testing was significantly higher than that of single-gene detection of APC and RASSF1A（χ2=23.30,15.33;P 0.05）. Conclusions The methylation consistency is favorable or well between tumour tissues and plasma in patients with ESCC.There is no significant superior in diagnosing ESCC with united testing of multiple tumor suppressor genes methylation in plasma than with single-gene detection.But the sensitivity of the former is better than the latter.（Chin J Lab Med, 2012,35:37-41） Key words: Esophageal neoplasms; Carcinoma，squamous cell; DNA methylation

Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe

Background Microarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets. Methods A set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface. Results This assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of target-selection-padlock probes and microarray. Conclusions The present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples.

Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is highly malignant and prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumour appearing is of clinical importance. At this point, no convenient method is available to determine the origin of these HCCs. Tissue samples were obtained from 19 patients and analysed for the promoter hypermethylation status of multiple tumour suppressor genes (p16, DAP-Kinase, MGMT, GSTP1, APC, RIZ1, SFRP1, SFRP2, SFRP5, RUNX3, and SOCS1) using methylation-specific PCR (MSP). Methylation status was used to determine tumour clonality. In each of the 19 cases, at least one tumour was recognised as having an aberrantly methylated gene. The frequency of the methylation in tumour tissue was 57.1% in p16, 2.4% in DAP-kinase, 23.8% in GSTP1, 90.5% in APC, 45.2% in RIZ1, 64.3% in SFRP1, 59.5% in SFRP2, 28.6% in SFRP5, 47.6% in RUNX3, and 54.8% in SOCS1, while in MGMT, no aberrant methylation was detected. The methylation status of these genes was assessed using MSP as being either positive or negative, and was used to determine the tumour clonality. The clonality of every tumour could be decided even with lesions that could not be judged by clinical diagnosis or by another molecular method (mt DNA mutation). Determining the methylation status of multiple genes in multicentric HCC was useful as a clonal marker and provided useful information for characterising the tumour. From our findings, multicentric HCCs tend to occur more independently than metastatically from the original tumour. Expanded study should be pursued further for a better understanding of the molecular mechanism of hepatocarcinogenesis.

Promoter Methylation Status of Multiple Genes in Uveal Melanoma

Aberrant promoter hypermethylation of CpG islands is thought to play an important role in the inactivation of tumor-suppressor genes (TSGs) in cancer. Studies of cutaneous melanoma have reported a high methylation rate for MGMT, DAPK, RAR-b2, and RASSF1A. In colon cancer, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G are commonly inactivated. The concomitant methylation of at least three of these genes may represent a distinct trait, the CpG island methylator phenotype (CIMP). The purpose of the present study was to investigate the role of epigenetic inactivation of multiple genes in uveal melanoma. Twenty samples of uveal melanoma were analyzed for the methylation status of nine candidate cancer-related genes: MGMT, DAPK, RAR-b2, RASSF1A, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G, using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. Methylation rates of the genes commonly inactivated in cutaneous melanoma were 70% for RASSFIA, 5% for MGMT and DAPK, and 0 for RAR-b2. The rates for the CIMP-related genes were 25% for RUNX3, 5% for NEUROG1 and CACNA1G, and 0 for SOCS-1 and IGF-2. None of the samples was CIMP-positive. In this study uveal melanoma was negative for CIMP, with hypermethylation of RASSF1A. The negative CIMP phenotype and frequent RASSF1A methylation in uveal melanoma is in accord with its known lack of BRAF mutations. Given that mutations in genes of the RAS pathway are rarely observed in uveal melanoma, epigenetic inactivation of RASSF1A may be an alternative mechanism of tumorigenesis. The low frequency of promoter methylation of TSGs commonly inactivated in cutaneous melanoma further stratifies the different tumorigenesis pathway in cutaneous and uveal melanoma.

In situ bisulfite modification of membrane-immobilized DNA for multiple methylation analysis

Increasing interest in DNA methylation has resulted in the recent development of array-based methods. However, these method require complex sample treatment processes, such as bisulfite modification in the liquid state of every sample. Here, we describe a microarray-based technology for interrogating DNA methylation status of multiple DNA samples at the same time, in which the DNA samples are first dotted on membranes and then treated with bisulfite directly. In this assay, plasmid pUC19 DNA is immobilized on nylon membranes and soaked in bisulfite reaction mixtures for 16 h to convert unmethylated Cs to Us, and methylated Cs remain unchanged. The probes for detecting the methylation patterns of CpG sites are hybridized with the converted DNA dotted on the membranes, and the signals are revealed by chemiluminescence of DIG-labeled probes. The experiments show that this method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This new method could be used as a convenient tool for detecting the methylation status of multiple genes for a large number of samples in the future.

Meth-DOP-PCR: an assay for the methylation profiling of trace amounts of DNA extracted from bodily fluids

Cancer cells release their DNA into the patient's bodily fluids and cancer-specific signatures can be recognized in the circulating DNA. The aberrant methylation of CpG-rich regions in gene promoter sequences is an early marker of cell transformation whose specificity and optimal sensitivity can be achieved by assessing the methylation status of multiple genes (‘methylation profiling'). Most of the current technologies for methylation analysis rely upon the combination of chemical conversion of the DNA and PCR analysis for the detection of methylated and unmethylated alleles. However, the small amount of circulating DNA, and its fragmentation, dramatically reduces the template DNA molecules making difficult the methylation profiling. To overcome this limitation, we have developed the Meth-DOP-PCR assay, a combination between a modified degenerate oligonucleotide primed PCR (DOP-PCR) and methylation-specific PCR (MSP), for the high-throughput methylation analysis of trace-amount of circulating DNA. We have demonstrated the concordance between Meth-DOP-PCR and MSP and shown the application of this technique for the methylation analysis of DNA extracted from the serum of lung cancer patients. We have estimated that through this procedure it is possible to obtain at least a 25-fold increase of the number of determinations allowing the methylation profiling from less than 1 ml of serum. Thus, Meth-DOP-PCR appears as a simple, cost-effective and efficient technique, for the development of novel methylation-based diagnostic assays.

Identification of 41 Novel Promoter-Associated CpG Islands Methylated in Leukemias.

DNA methylation within promoter-associated CpG islands is a well-recognized mechanism of gene silencing and plays an important role in the development of malignancies. CpG dinucleotides in human DNA are methylated at 5′-cytosine with the exception of areas with dense concentration of CpGs (CpG islands) located in gene promoter regions. In cancer cells, methylation of CpG islands in promoter regions of tumor suppressor genes is a frequent epigenetic change with a gene-silencing effect analogous to inactivating mutations. Methylation profiling can identify biologically and clinically distinct tumor subgroups by mapping the methylation status of multiple genes, and reports in AML and ALL suggest associations between methylation and poor prognosis. Identification of methylated CpG islands can shed new light on the biology of leukemia. We used Methylated CpG Island Amplification coupled with Representative Difference Analysis (MCA-RDA) as a genome-wide screen for promoter-associated CpG islands methylated in leukemic and/or myeloproliferative cell lines and primary malignant cells, but unmethylated in blood cells from normal controls. We identified 51 unique promoter-associated CpG islands in 321 sequenced clones recovered by MCA-RDA. Forty-one CpG islands belonged to known genes, and 10 to annotated mRNAs. Of the genes with known function, 8 are involved in signaling, 7 in transcription, 3 in dephosphorylation, 2 in oxido-reductive processes, 2 in NO synthesis, 2 in adhesion, 2 in solute transport, and 2 in DNA replication. Seven out of the 51 genes were previously reported as methylated in cancer or leukemia (CDH13, HLA-B, HLA-C, PGR, SCGB3A1, SLC26A4, TERT), thus validating the MCA-RDA approach. Of the 41 new hypermethylated CpG islands recovered, 20 corresponded to genes of known function. Published data infer an association with cancer for 10 of these genes (CTDSPL, ECGF1, EDG4, FOXD2, NOR1, NOS3, OLIG2, SLC16A1, TLE1, WNT5B), and no reports were found for the other 10 genes (CNR1, FADS, FBXW3, FGD1, NPM2, P518, PDE4DIP, SNCB, TCEA3, VENTX2). To further validate our findings we are assessing the methylation status of these genes by bisulfite pyrosequencing. Analyses of the bone marrow samples from AML, ALL, CML and MDS patients are ongoing. Our preliminary data confirm methylation of H-cadherin precursor (CDH13), progesterone receptor (PGR) in AML and ALL and cannabinoid receptor 1 (CNR1) in ALL (Table). In conclusion, MCA-RDA identified methylation of 41 new and 10 previously reported promoter-associated CpG islands in leukemia. Functional studies of these may shed new light on the biology of leukemias, and these genes may be useful for methylation profiling of leukemias for prognosis and response to treatment.Promoter CpG Island MethylationGeneAMLALLCDH135/2322%12/1963%PGR A Isoform11/2250%12/1867%PGR B Isoform17/2471%5/1338%CNR10/240%3/1817%Methylation levels over 10% for CDH13 and PGR and over 25% for CNR1 were scored as positive.

High-throughput method for detecting DNA methylation

Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.

Promoter methylation status of multiple genes in brain metastases of solid tumors

The aberrant methylation of the CpG island promoter regions acquired by tumor cells is one mechanism for loss of gene function. The high methylation rate for RB1 and death-associated protein-kinase gene (DAP-kinase) (60 and 90%, respectively) previously found in brain metastases suggests this mechanism could be non-randomly associated to tumor progression and metastasis. Thus, in addition to these two genes, we determined the methylation status of the genes p16INK4a, glutathione S-transferase P1 (GSTP1), O6-methylguanine DNA methyltransferase (MGMT), thrombospondin-1 (THBS1), p14ARF, TP53, p73, and tissue inhibitor of metalloproteinase 3 (TIMP-3), in 18 brain metastases of solid tumors, with methylation specific PCR. The metastases were derived from malignant melanoma (three cases), lung carcinoma (six cases), breast carcinoma (three cases), ovarian carcinoma (two cases) and one each from colon, kidney, bladder and undifferentiated carcinoma. We detected methylation levels in the tumor samples of 83% in p16INK4a, 72% in DAP-kinase, 56% in THBS1, 50% in RB1, 39% in MGMT, 33% in GSTP1 and p14ARF each, 22% in p73 and TIMP-3 each, and 11% in TP53. The methylation index (number of genes methylated/number of genes tested) varied between 0.1 and 0.6, with an average of 0.42, indicating that a high grade of gene methylation accumulates parallel to the tumor metastasis process. Our data suggest an important role for gene methylation in the development of brain metastases, primarily involving epigenetic silencing of DAP-kinase, THBS1 and the cell-cycle regulators RB1/p16INK4a.