Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation

Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island.

BACKGROUND: The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. METHODS: Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). RESULTS: Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (<=50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. CONCLUSIONS: Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon(R)), providing further support for the clinical relevance of this biomarker

Evaluation of an epigenetic assay for predicting repeat prostate biopsy outcome in African American men

OBJECTIVE: To evaluate an epigenetic assay performed on tissue from negative prostate biopsies in a group of African American (AA) men undergoing repeat biopsy, and to compare accuracy for predicting repeat biopsy outcome to prior studies conducted in predominantly Caucasian populations. MATERIALS AND METHODS: The study population consisted of 211 AA men from 7 urology centers across the United States; all of whom were undergoing 12-core transrectal ultrasound-guided repeat biopsy within 30 months from a negative index biopsy. All biopsy cores from the negative index biopsy were profiled for the epigenetic biomarkers GSTP1, APC, and RASSF1 using ConfirmMDx for Prostate Cancer (MDxHealth, Irvine, CA). RESULTS: Upon repeat biopsy, 130 of 211 subjects (62%) had no prostate cancer (PCa) detected and 81 of 211 (38%) were diagnosed with PCa. Of the subjects with PCa, 54 (67%) were diagnosed with Gleason score (GS) = 7 disease. For detection of PCa at repeat biopsy, ConfirmMDx sensitivity was 74.1% and specificity was 60.0%, equivalent to prior studies (P = .235 and .697, respectively). For detection of GS >= 7 PCa, sensitivity was 78% and specificity was 53%. The negative predictive values for detection of all PCa and GS >= 7 PCa were 78.8% and 94.2%, respectively. CONCLUSION: In this group of AA men, we successfully validated an epigenetic assay to assess the need for repeat biopsy. Results were consistent with previous studies from predominantly Caucasian populations. Therefore, the ConfirmMDx assay is a useful tool for risk stratification of AA men who had an initial negative biopsy

CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients

<p>Abstract</p> <p>Background</p> <p>Various biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy.</p> <p>Methods</p> <p>Quantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival.</p> <p>Results</p> <p>Of the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).</p> <p>Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010).</p> <p>Conclusions</p> <p>CDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.</p

Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon

In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR–DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR–DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues

Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia

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Characterization of DNA methylation for biomarkers in breast and colon cancer

Diese Arbeit umfasst die Charakterisierung der DNA-Methylierung von Biomarkern für Brust- und Darmkrebs auf mikrodissektiertem Gewebe, sowie der Identifizierung neuer prognostischer Marker für Brustkrebs. Im Rahmen der Arbeit wurden dabei Methoden für multiplexierte PCRs von sehr geringen DNA Mengen aus mikrodissektierten Zellen etabliert und optimiert. Es wird gezeigt, dass sich die DNA-Methylierung in heterogenen Tumorproben von der in Epithel- und Stromazellen unterscheidet. Die Analyse der Methylierung des für Brustkrebs prognostischen Biomarkers PITX2 auf Tumorepithelzellen könnte zu einer Verbesserung der prognostischen Aussagekraft beitragen. Die etablierte multiplexierte PCR ermöglicht die Analyse der DNA-Methylierung von PITX2 und acht weiteren Biomarkern aus einer einzigen mikrodissektierten Probe. Eine weitere multiplexierte Mikrodissektionsstudie untersucht unter anderem die DNA-Methylierung von sieben Regionen des für Darmkrebs diagnostischen Biomarkers SEPT9 in Tumor und Tumor benachbarten Epithel- und Stromazellen. Die Ergebnisse zeigen, dass die Methylierung in den untersuchten Regionen nicht homogen verteilt ist: CpG Inseln im intragenischen Bereich zeigen deutlich stärkere differentielle Methylierung zwischen den Zelltypen als solche im Bereich des Transkriptionsstarts. Eine quantitative direkte Bisulfit-Sequenzierungsstudie zur Identifizierung von prognostischen Markern in 162 Brusttumoren mit 202 potentiellen Markerkandidaten resultiert in einem DNA-Methylierungs Biomarker im Promotor des Gens CDO1. Dieser zeigte in den untersuchten Brustkrebsproben von adjuvant mit Anthrazyklin behandelten Patientinnen signifikante prognostische Eigenschaften.This thesis describes the characterisation of DNA methylation for known breast and colon cancer biomarkers on microdissected cells, as well as identification DNA methylation within seven different regions of the diagnostic biomarker for colon cancer, SEPT9, was investigated in a study of microdissected colon tumor epithelial and stromal cells along with adjacent cells. The results indicate that DNA methylation at different regions of SEPT9 is not homogenous: CpG Islands in intragenic regions of SEPT9 show significantly higher differential DNA methylation levels between the cell types compared to regions close to the transcription start site. of a potential novel prognostic DNA methylation marker for breast cancer. In order to analyse DNA methylation in different regions of a limited number of microdissected cells, a method for multiplexing PCRs was established. The results of this microdissection study in breast tumors showed that the DNA methylation of a biomarker can differ in a heterogeneous sample compared to microdissected epithelial and stromal cells from the same tumor. Evidence is shown that the DNA methylation measurement of the known prognostic biomarker, PITX2, in tumor epithelial cells may even contribute to enhancement of the prognostic power of PITX2. Lastly, a quantitative bisulfite sequencing study was conducted to analyze the DNA methylation of 202 potential biomarker candidates in a retrospective cohort of 162 breast cancer patients. The DNA methylation of CDO1 methylation was shown to be a potential strong predictor for distant metastasis in the cohort of breast cancer patients who received adjuvant anthracycline-based chemotherapy

Analytical validation of an mRNA-based urine test to predict the presence of high-grade prostate cancer

Abstract Background The challenge for prostate cancer (PCa) diagnosis is to improve the ability to distinguish indolent from aggressive PCa. Increased urinary levels of Distal-less Homeobox 1 (DLX1) and Homeobox C6 (HOXC6) mRNA have been associated with high-grade PCa (Gleason Score ≥7). Reverse transcriptase PCR (RT-PCR)-assays for the quantification of DLX1 and HOXC6 mRNA levels were developed, and clinical validation showed that these urinary markers can significantly improve accuracy for detection of high-grade PCa at biopsy. The aim of this study was to validate the analytical performance of these mRNA-based assays, including specimen stability and analytical sensitivity, specificity, precision, repeatability and reproducibility. Methods Analytical validation of the RT-PCR assays for DLX1, HOXC6 and KLK3 was performed using in vitro transcribed (IVT) specimens of the target genes, covering all aspects of the analytical method including assay sensitivity, specificity, linearity, precision, repeatability and reproducibility using pre-specified acceptance criteria. To confirm that the mRNA assays are sufficient robust for use in clinical routine laboratories, the performance characteristics were verified in an independent laboratory using post-DRE collected urine samples from 101 men scheduled for prostate biopsy. Results A stabilization buffer, developed for urine preservation under standard pre-processing conditions, makes sample shipment from clinics to laboratories easily feasible. The mRNA in urine samples, preserved in this stabilization buffer, is stable at room temperature up to 5 days from collection, resulting in 100% evaluable rate. The long term stability of the mRNAs in the buffer was shown by similar clinical performances using RNA values obtained immediately after urine collection (area under curve (AUC) 0.72(95% CI: 0.61–0.83)) and after 1-year storage (AUC 0.71 (95% CI: 0.60–0.81)). Test performance was not compromised by most common inhibitors and bacterial strains found in urine. However, an inhibitory effect of hemoglobin was observed. All precision, reproducibility, instrument and inter-laboratory variation data obtained for the mRNA-based assays met the pre-specified acceptance criteria of a standard deviation less than or equal to 0.5 crossing point. The analysis of 99 whole urine samples at two laboratories indicated a very strong positive correlation (r = 0.997, P < 0.001). The test outcome in terms of absolute difference in likelihood for high-grade PCa upon biopsy was less than 2% between the two sites. Conclusions This study illustrates the robustness of the mRNA assays, enabling testing in clinical routine laboratories and molecular pathology laboratories where the here described automated RNA extraction and PCR platforms are available