Method development for quantitative methylation analysis by direct bisulfite sequencing, raw data processing and analysis of the Human Epigenome Project

Epigenetic deals with flexible biochemical information layers that lie on top of the relatively stable DNA sequence and are involved in control of the structure and functionality of the DNA. One of the most easily analyzed epigenetic layers is DNA methylation. The first part of this work describes the development and assessment of new algorithms enabling methylation quantification by interpretation of sequencing electropherogram data from direct bisulfite sequencing. It is shown that the use of the algorithms on data from PCR products is a suitable replacement for subcloning and sequencing of about 10 subclones - a prerequisite for efficient high throughput DNA methylation studies by direct sequencing, such as carried out in the Human Epigenome Project (HEP). The second part demonstrates the possibility to compensate for artifacts and signal echos in raw data from direct bisulfite sequencing using a deconvolution algorithm. In the third part of this thesis the data of the HEP is analyzed. The HEP is the first large-scale project providing high resolution methylation data in 12 healthy human tissue types on 3 chromosomes analyzed, with a view to answering biological questions. It is shown that differential methylation between healthy tissues is a common phenomenon - especially in conserved non coding sequences, how CpG density and proximity to functional genomic sites influence the methylation profile and in how far CpGs tend to be organized in co-methylated blocks.Epigenetik beschäftigt sich mit dynamischen biochemischen Informationsebenen, welche die im Vergleich dazu relativ stabile DNS beeinflussen und eine Rolle bei der Kontrolle der Struktur und Funktion der DNS spielt. DNS Methylierung ist eine der am besten untersuchbaren epigenetischen Ebenen. Diese Arbeit beschreibt im ersten Teil die Entwicklung eines neuen Algorithmus, der quantitative Methylierungsmessung auf der Grundlage von Elektropherogramm Daten aus der direkten Sequenzierung von PCR Produkten von Bisulfit behandelter DNS ermöglicht. Es wird gezeigt, daß die Verwendung des Algorithmus mit Daten von PCR Produkten eine brauchbare Alternative zu Subklonierung und Sequenzierung von ca. zehn Subklonen ist und damit effiziente DNS Methylierungsstudien durch Hochdurchsatzsequenzierung ermöglicht. Der zweite Teil der Arbeit beschreibt die Möglichkeit mit Hilfe eines Dekonvolutions Algorithmus Artefakte und Signal Echos in Sequenzierungsrohdaten zu kompensieren. Der dritte Teil behandelt die Analyse und die biologische Erkenntnisse aus den Daten des HEP, dem ersten hochauflösenden Methylierungsdatensatz für drei Chromosomen in zwölf Geweben. Es wird gezeigt, daß differentielle Methylierung zwischen gesunden Geweben weit verbreitet ist, welchen Einfluß CpG Dichte und Nachbarschaft zu funktionalen genomischen Bereichen auf das DNS Methylierungsprofil haben und in wieweit benachbarte CpGs dazu tendieren, sich in comethylierten Einheiten zu organisieren

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

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

DNA Methylation Profiling of the Human Major Histocompatibility Complex: A Pilot Study for the Human Epigenome Project

The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine–guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

Methodenentwicklung für die quantitative Methylierungsanalyse durch direkte Bisulfitsequenzierung, Rohdatenprozessierung und Analyse des Humanen Epigenom-Projektes

Epigenetic deals with flexible biochemical information layers that lie on top of the relatively stable DNA sequence and are involved in control of the structure and functionality of the DNA. One of the most easily analyzed epigenetic layers is DNA methylation. The first part of this work describes the development and assessment of new algorithms enabling methylation quantification by interpretation of sequencing electropherogram data from direct bisulfite sequencing. It is shown that the use of the algorithms on data from PCR products is a suitable replacement for subcloning and sequencing of about 10 subclones - a prerequisite for efficient high throughput DNA methylation studies by direct sequencing, such as carried out in the Human Epigenome Project (HEP). The second part demonstrates the possibility to compensate for artifacts and signal echos in raw data from direct bisulfite sequencing using a deconvolution algorithm. In the third part of this thesis the data of the HEP is analyzed. The HEP is the first large-scale project providing high resolution methylation data in 12 healthy human tissue types on 3 chromosomes analyzed, with a view to answering biological questions. It is shown that differential methylation between healthy tissues is a common phenomenon - especially in conserved non coding sequences, how CpG density and proximity to functional genomic sites influence the methylation profile and in how far CpGs tend to be organized in co-methylated blocks.Epigenetik beschäftigt sich mit dynamischen biochemischen Informationsebenen, welche die im Vergleich dazu relativ stabile DNS beeinflussen und eine Rolle bei der Kontrolle der Struktur und Funktion der DNS spielt. DNS Methylierung ist eine der am besten untersuchbaren epigenetischen Ebenen. Diese Arbeit beschreibt im ersten Teil die Entwicklung eines neuen Algorithmus, der quantitative Methylierungsmessung auf der Grundlage von Elektropherogramm Daten aus der direkten Sequenzierung von PCR Produkten von Bisulfit behandelter DNS ermöglicht. Es wird gezeigt, daß die Verwendung des Algorithmus mit Daten von PCR Produkten eine brauchbare Alternative zu Subklonierung und Sequenzierung von ca. zehn Subklonen ist und damit effiziente DNS Methylierungsstudien durch Hochdurchsatzsequenzierung ermöglicht. Der zweite Teil der Arbeit beschreibt die Möglichkeit mit Hilfe eines Dekonvolutions Algorithmus Artefakte und Signal Echos in Sequenzierungsrohdaten zu kompensieren. Der dritte Teil behandelt die Analyse und die biologische Erkenntnisse aus den Daten des HEP, dem ersten hochauflösenden Methylierungsdatensatz für drei Chromosomen in zwölf Geweben. Es wird gezeigt, daß differentielle Methylierung zwischen gesunden Geweben weit verbreitet ist, welchen Einfluß CpG Dichte und Nachbarschaft zu funktionalen genomischen Bereichen auf das DNS Methylierungsprofil haben und in wieweit benachbarte CpGs dazu tendieren, sich in comethylierten Einheiten zu organisieren

Adjoint-based optimization of sound reinforcement including non-uniform flow

The determination of optimal geometric arrangements and electronic drives of loudspeaker arrays in sound reinforcement applications is an ill-posed inverse problem. This paper introduces an innovative method to determine complex driving functions, also considering complex environmental conditions. As an alternative to common frequency domain methods, the authors present an adjoint-based approach in the time domain: Acoustic sources are optimized in order to generate a given target sound field. Instead of the Helmholtz equation, the full non-linear Euler equations are considered. This enables an easier treatment of non-uniform flow and boundary conditions. As proof of concept, a circular and a linear monopole array are examined. For the latter, the environmental conditions include wind and thermal stratification. For all examples, the method is able to provide appropriate driving functions.DFG, 393106680, Optimale Schallfelderzeugung für Beschallungsaufgaben im Zeit- und Frequenzbereic