Dna Mutation/Methylation Screening Method for Colon Cancer Screening

Cancer is a result of unregulated cell growth. For all types of cancer currently studied, the transition from a healthy cell to a malignant tumor cell is a step-by-step process which requires mutation in at least several oncogenes and tumor suppressor genes together. Another cancer early event is DNA methylation. Cancer-related DNA methylation focuses on promoter hypermethylation of the certain genes. The DNA mutation and methylation profile can serve as biomarkers for diagnosing early stage of cancer. Colorectal cancer is the third most common cancer type in the United States and has been well studied. The essential mechanism of cancer development is becomeing clear, so there are more approaches to diagnose early stage cancer and improve cancer treatment, which benefits colorectal cancer screening in recent years. The current mutation/methylation detection techniques generally have two major categories which rely on the 1) physical property of double strand DNA or 2) enzyme selectivity to survey the target sequence. Chapters I and III summarize the major methods used in the present DNA mutation and methylation analysis. High Resolution Melting (HRM) is a simple, PCR-based method for detecting DNA sequence variation by measuring the melting temperature of a DNA duplex. In Chapter II, a robust and lower cost HRM assay for screening P53 and Kras mutations is discussed. In Chapter IV we developed Probe Enrichment Mutation/Methylation-High Resolution Melting (PEMM-HRM) assay. PEMM-HRM analysis is a simple and high sensitive post PCR technique which can be used for high throughput mutation scanning, genotyping and methylation analysis. PEMM-HRM analysis with enhanced sensitivity and specificity can have broad applications in clinical research. In chapter V, We studied adenylosuccinate lyase deficiency, which is a defect of purine metabolism. We developed a method combining ESI-MS with solid-phase extraction to detect succinyladenosine (SA) and succinylamino-imidazolecarboxamide riboside (SAICAr) of patients with ade

Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus

The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (∼90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression

Dna Mutation/Methylation Screening Method for Colon Cancer Screening

Cancer is a result of unregulated cell growth. For all types of cancer currently studied, the transition from a healthy cell to a malignant tumor cell is a step-by-step process which requires mutation in at least several oncogenes and tumor suppressor genes together. Another cancer early event is DNA methylation. Cancer-related DNA methylation focuses on promoter hypermethylation of the certain genes. The DNA mutation and methylation profile can serve as biomarkers for diagnosing early stage of cancer. Colorectal cancer is the third most common cancer type in the United States and has been well studied. The essential mechanism of cancer development is becomeing clear, so there are more approaches to diagnose early stage cancer and improve cancer treatment, which benefits colorectal cancer screening in recent years. The current mutation/methylation detection techniques generally have two major categories which rely on the 1) physical property of double strand DNA or 2) enzyme selectivity to survey the target sequence. Chapters I and III summarize the major methods used in the present DNA mutation and methylation analysis. High Resolution Melting (HRM) is a simple, PCR-based method for detecting DNA sequence variation by measuring the melting temperature of a DNA duplex. In Chapter II, a robust and lower cost HRM assay for screening P53 and Kras mutations is discussed. In Chapter IV we developed Probe Enrichment Mutation/Methylation-High Resolution Melting (PEMM-HRM) assay. PEMM-HRM analysis is a simple and high sensitive post PCR technique which can be used for high throughput mutation scanning, genotyping and methylation analysis. PEMM-HRM analysis with enhanced sensitivity and specificity can have broad applications in clinical research. In chapter V, We studied adenylosuccinate lyase deficiency, which is a defect of purine metabolism. We developed a method combining ESI-MS with solid-phase extraction to detect succinyladenosine (SA) and succinylamino-imidazolecarboxamide riboside (SAICAr) of patients with ade

DNA hydroxymethylation levels are altered in blood cells from Down syndrome persons enrolled in the MARK-AGE project

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the ageing process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work we investigated the levels of 5-hydroxymethylcytosine (5hmC) and of the TET dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in ageing. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease of 5hmC, TET1 and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS

Methylation status of Vitamin D receptor gene promoter in benign and malignant adrenal tumors

We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5' regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesi

Gene regulation and epigenotype in Friedreich's ataxia

Friedreich??????s ataxia (FRDA) is known to be provoked by an abnormal GAA-repeat expansion located in the first intron of the FXN gene. As a result of the GAA expansion, patients exhibit low levels of FXN mRNA, leading to FRDA. Here, via chromatin immunoprecipitation (ChIP), the presence of a RNA pol II transcriptional pausing site at exon 1 of the FXN gene was demonstrated. At this site, FRDA EBVcell lines exhibited elevated levels of the negative elongation factor NELF-E depending on the presence of a GAA repeat expansion compared to controls. This site may represent a rate-limiting step for FXN transcription and consequently provide a means to modify transcription levels in FRDA. Moreover, RNA pol II pausing site binding factors, such as NELF-E, were influenced by Nicotinamide treatment, a HDAC class III inhibitor. Therefore, factors sensitive to chromatin changes may influence the regulation of RNA pol II pausing and also balance otherwise positive chromatin changes. This new finding could explain the relatively minor effects of different drug approaches to up-regulate this gene. Furthermore, CTCF and the histone demethylase LSD1 were also found to be located at the FXN pausing site. Results suggest a function for LSD1 in demethylating H3K4me2 at the pausing site and potentially also in demethylating H3K9me3 in the case of frequently transcribed expanded GAA repeats. Therefore, LSD1 might play a crucial role in preventing heterochromatinisation of a euchromatic gene. Using primary transcript RNA-FISH, a delay in RNA pol II release from the pausing site and furthermore a dramatic loss of RNA pol II elongation in the presence of expanded GAA repeats was seen. The identified and characterised transcriptional pausing site at FXN is likely to play a repressive role and participates in the pathogenesis of FRDA.Imperial Users onl

A novel epigenetic AML1-ETO/THAP10/miR-383 mini-circuitry contributes to t(8;21) leukaemogenesis

DNA methylation patterns are frequently deregulated in t(8;21) acute myeloid leukaemia (AML), but little is known of the mechanisms by which specific gene sets become aberrantly methylated. Here, we found that the promoter DNA methylation signature of t(8;21)(+) AML blasts differs from that of t(8;21)(-) AMLs. This study demonstrated that a novel hypermethylated zinc finger-containing protein, THAP10, is a target gene and can be epigenetically suppressed by AML1-ETO at the transcriptional level in t(8;21) AML. Our findings also show that THAP10 is a bona fide target of miR-383 that can be epigenetically activated by the AML1-ETO recruiting co-activator p300. In this study, we demonstrated that epigenetic suppression of THAP10 is the mechanistic link between AML1-ETO fusion proteins and tyrosine kinase cascades. In addition, we showed that THAP10 is a nuclear protein that inhibits myeloid proliferation and promotes differentiation both in vitro and in vivo Altogether, our results revealed an unexpected and important epigenetic mini-circuit of AML1-ETO/THAP10/miR-383 in t(8;21) AML, in which epigenetic suppression of THAP10 predicts a poor clinical outcome and represents a novel therapeutic target

A subset of methylated CpG sites differentiate psoriatic from normal skin.

Psoriasis is a chronic inflammatory immune-mediated disorder affecting the skin and other organs including joints. Over 1,300 transcripts are altered in psoriatic involved skin compared with normal skin. However, to our knowledge, global epigenetic profiling of psoriatic skin is previously unreported. Here, we describe a genome-wide study of altered CpG methylation in psoriatic skin. We determined the methylation levels at 27,578 CpG sites in skin samples from individuals with psoriasis (12 involved, 8 uninvolved) and 10 unaffected individuals. CpG methylation of involved skin differed from normal skin at 1,108 sites. Twelve mapped to the epidermal differentiation complex, upstream or within genes that are highly upregulated in psoriasis. Hierarchical clustering of 50 of the top differentially methylated (DM) sites separated psoriatic from normal skin samples with uninvolved skin exhibiting intermediate methylation. CpG sites where methylation was correlated with gene expression are reported. Sites with inverse correlations between methylation and nearby gene expression include those of KYNU, OAS2, S100A12, and SERPINB3, whose strong transcriptional upregulation is an important discriminator of psoriasis. Pyrosequencing of bisulfite-treated DNA from skin biopsies at three DM loci confirmed earlier findings and revealed reversion of methylation levels toward the non-psoriatic state after 1 month of anti-TNF-α therapy