Altered microRNA and target gene expression related to Tetralogy of Fallot

MicroRNAs (miRNAs) play an important role in guiding development and maintaining function of the human heart. Dysregulation of miRNAs has been linked to various congenital heart diseases including Tetralogy of Fallot (TOF), which represents the most common cyanotic heart malformation in humans. Several studies have identified dysregulated miRNAs in right ventricular (RV) tissues of TOF patients. In this study, we profiled genome-wide the whole transcriptome and analyzed the relationship of miRNAs and mRNAs of RV tissues of a homogeneous group of 22 non-syndromic TOF patients. Observed profiles were compared to profiles obtained from right and left ventricular tissue of normal hearts. To reduce the commonly observed large list of predicted target genes of dysregulated miRNAs, we applied a stringent target prediction pipeline integrating probabilities for miRNA-mRNA interaction. The final list of disease-related miRNA-mRNA pairs comprises novel as well as known miRNAs including miR-1 and miR-133, which are essential to cardiac development and function by regulating KCNJ2, FBN2, SLC38A3 and TNNI1. Overall, our study provides additional insights into post-transcriptional gene regulation of malformed hearts of TOF patients

Accuracy of a magnetic resonance imaging‐based 3D printed stereotactic brain biopsy device in dogs

Background: Brain biopsy of intracranial lesions is often necessary to determine specific therapy. The cost of the currently used stereotactic rigid frame and optical tracking systems for brain biopsy in dogs is often prohibitive or accuracy is not sufficient for all types of lesion. Objectives: To evaluate the application accuracy of an inexpensive magnetic resonance imaging‐based personalized, 3D printed brain biopsy device. Animals: Twenty‐two dog heads from cadavers were separated into 2 groups according to body weight (20 kg). Methods: Experimental study. Two target points in each cadaver head were used (target point 1: caudate nucleus, target point 2: piriform lobe). Comparison between groups was performed using the independent Student's t test or the nonparametric Mann‐Whitney U Test. Results: The total median target point deviation was 0.83 mm (range 0.09‐2.76 mm). The separate median target point deviations for target points 1 and 2 in all dogs were 0.57 mm (range: 0.09‐1.25 mm) and 0.85 mm (range: 0.14‐2.76 mm), respectively. Conclusion and Clinical Importance: This magnetic resonance imaging‐based 3D printed stereotactic brain biopsy device achieved an application accuracy that was better than the accuracy of most brain biopsy systems that are currently used in veterinary medicine. The device can be applied to every size and shape of skull and allows precise positioning of brain biopsy needles in dogs

A Comparative Study of Automatic Localization Algorithms for Spherical Markers within 3D MRI Data

Localization of features and structures in images is an important task in medical image-processing. Characteristic structures and features are used in diagnostics and surgery planning for spatial adjustments of the volumetric data, including image registration or localization of bone-anchors and fiducials. Since this task is highly recurrent, a fast, reliable and automated approach without human interaction and parameter adjustment is of high interest. In this paper we propose and compare four image processing pipelines, including algorithms for automatic detection and localization of spherical features within 3D MRI data. We developed a convolution based method as well as algorithms based on connected-components labeling and analysis and the circular Hough-transform. A blob detection related approach, analyzing the Hessian determinant, was examined. Furthermore, we introduce a novel spherical MRI-marker design. In combination with the proposed algorithms and pipelines, this allows the detection and spatial localization, including the direction, of fiducials and bone-anchors

Case Report: Clinical Use of a Patient-Individual Magnetic Resonance Imaging-Based Stereotactic Navigation Device for Brain Biopsies in Three Dogs

Three-dimensional (3D) printing techniques for patient-individual medicine has found its way into veterinary neurosurgery. Because of the high accuracy of 3D printed specific neurosurgical navigation devices, it seems to be a safe and reliable option to use patient- individual constructions for sampling brain tissue. Due to the complexity and vulnerability of the brain a particularly precise and safe procedure is required. In a recent cadaver study a better accuracy for the 3D printed MRI-based patient individual stereotactic brain biopsy device for dogs is determined compared to the accuracies of other biopsy systems which are currently used in veterinary medicine. This case report describes the clinical use of this 3D printed MRI-based patient individual brain biopsy device for brain sampling in three dogs. The system was characterized by a simple handling. Furthermore, it was an effective and reliable tool to gain diagnostic brain biopsy samples in dogs with no significant side effects

Induced pluripotent stem cells of patients with Tetralogy of Fallot reveal transcriptional alterations in cardiomyocyte differentiation

Patient-specific induced pluripotent stem cells (ps-iPSCs) and their differentiated cell types are a powerful model system to gain insight into mechanisms driving early developmental and disease-associated regulatory networks. In this study, we use ps-iPSCs to gain insights into Tetralogy of Fallot (TOF), which represents the most common cyanotic heart defect in humans. iPSCs were generated and further differentiated to cardiomyocytes (CMs) using standard methods from two well-characterized TOF patients and their healthy relatives serving as controls. Patient-specific expression patterns and genetic variability were investigated using whole genome and transcriptome sequencing data. We first studied the clonal mutational burden of the derived iPSCs. In two out of three iPSC lines of patient TOF-01, we found a somatic mutation in the DNA-binding domain of tumor suppressor P53, which was not observed in the genomic DNA from blood. Further characterization of this mutation showed its functional impact. For patient TOF-02, potential disease-relevant differential gene expression between and across cardiac differentiation was shown. Here, clear differences at the later stages of differentiation could be observed between CMs of the patient and its controls. Overall, this study provides first insights into the complex molecular mechanisms underlying iPSC-derived cardiomyocyte differentiation and its transcriptional alterations in TOF

Accuracy of a magnetic resonance imaging-based 3D printed stereotactic brain biopsy device in dogs

Background Brain biopsy of intracranial lesions is often necessary to determine specific therapy. The cost of the currently used stereotactic rigid frame and optical tracking systems for brain biopsy in dogs is often prohibitive or accuracy is not sufficient for all types of lesion. Objectives To evaluate the application accuracy of an inexpensive magnetic resonance imaging-based personalized, 3D printed brain biopsy device. Animals Twenty-two dog heads from cadavers were separated into 2 groups according to body weight (20 kg). Methods Experimental study. Two target points in each cadaver head were used (target point 1: caudate nucleus, target point 2: piriform lobe). Comparison between groups was performed using the independent Student's t test or the nonparametric Mann-Whitney U Test. Results The total median target point deviation was 0.83 mm (range 0.09-2.76 mm). The separate median target point deviations for target points 1 and 2 in all dogs were 0.57 mm (range: 0.09-1.25 mm) and 0.85 mm (range: 0.14-2.76 mm), respectively. Conclusion and Clinical Importance This magnetic resonance imaging-based 3D printed stereotactic brain biopsy device achieved an application accuracy that was better than the accuracy of most brain biopsy systems that are currently used in veterinary medicine. The device can be applied to every size and shape of skull and allows precise positioning of brain biopsy needles in dogs

The Cardiac Transcription Network Modulated by Gata4, Mef2a, Nkx2.5, Srf, Histone Modifications, and MicroRNAs

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA–binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi–mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA–binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders

Computergestützte Analyse von Next-Generation-Sequenzierungsdaten hinsichtlich der Herzfunktion und -erkrankung

Over the past years, there has been a fundamental shift away from the application of semi-automated Sanger sequencing for genome analysis to so- called next-generation sequencing (NGS). The main advantage offered by NGS is the ability to sequence millions of DNA fragments in a very short time scale. There is a wide range of NGS applications, rapidly developing, making the computational analysis of their associated datasets very challenging. For gene expression analysis microarrays are more and more being replaced by sequenced- based methods, which can identify and quantify coding and non-coding transcripts without prior knowledge. Genome sequencing either at a whole or for particular sequences (targeted resequencing) enable the identification of genomic variations at a broad scale. This thesis approaches computational challenges of NGS technologies applied for targeted DNA resequencing, sequencing of expressed mRNAs (RNA-seq) and miRNAs (miRNA-seq) as well as the identification of protein-DNA interactions such as transcription factor binding sites or chromatin histone marks (ChIP-seq). Experimental datasets generated within the group as well as publicly available were used to develop novel computational approaches and bioinformatics tools for the analysis of NGS datasets and eventually answer biological questions regarding cardiac function and disease. A first study is focused on the combinatorial regulation of cardiac DNA-binding transcription factors (ChIP-seq of Srf) influenced by histone modifications (histone 3 acetylation) and regulatory miRNAs (miRNA- seq). As published in PLoS Genetics in 2011 these different levels regulating mRNA profiles have a high degree of interdependency and the potential to modulate each other. For example the effect of Srf binding is significantly influenced by the co-occurrence of histone 3 acetylation marks. Furthermore, differential expression of miRNAs can explain 45% of all differentially expressed mRNAs in Srf knockdown and approximately 50% of differential expression is driven by other secondary effects. Thus, to obtain a full picture of the regulatory transcription network underlying cardiomyocyte function, the different modulators need to be viewed in context to each other. Within this project the tool MicroRazerS was developed (published in Bioinformatics 2010). MicroRazerS is optimized for mapping small RNAs such as miRNAs or other small non-coding RNAs onto a reference genome. It is characterized by a higher sensitivity and an at least comparable speed to other short read mapping tools. The results suggest that MicroRazerS can substantially facilitate the profiling and discovery of miRNAs obtained from high-throughput sequencing. A second project aimed to identify the genetic basis of Tetralogy of Fallot (TOF). TOF accounts for up to 10% of all congenital heart disease, which are the most common birth defect in human. This study shows first time that TOF is an oligogenic disorder. We performed a multilevel study including targeted resequencing of over 1,000 heart- and muscle-relevant genes and miRNAs in TOF cases, parents and controls as well as whole transcriptome and miRNome analysis in TOF cases and healthy unaffected individuals using NGS techniques (87 samples). Genes were assessed according to the presence of deleterious variations and their rate of mutation in TOF subjects compared to healthy controls (200 cases). A set of 16 TOF genes was identified of which on average four genes per TOF subject are mutated and which discriminate TOF cases from controls. The computational approach developed within this study opens a new perspective for the analysis of oligo- or multigenic disorders in general.Im Bereich der Genanalyse hat es in den vergangenen Jahren eine wesentliche Abkehr von der Anwendung der halbautomatisierten Sanger-Sequenzierung hin zur sogenannten Next-Generation-Sequenzierung (NGS) gegeben. Der Hauptvorteil dieser NGS-Methoden liegt vor allem in der Fähigkeit Millionen von DNS- Fragmenten in sehr kurzer Zeit zu sequenzieren. Insgesamt gibt es eine breite Palette von NGS-Anwendungen, die sich schnell weiterentwickeln, was die computergestützte Analyse der damit verbundenen Datenmengen sehr anspruchsvoll macht. In der Genexpressionsanalyse werden die früher herkömmlichen Microarrays mehr und mehr durch sequenzbasierte Methoden ersetzt, die kodierenden und nicht-kodierenden Transkripte ohne deren vorherige Kenntnis identifizieren und quantifizieren können. Die Sequenzierung eines ganzen Genoms oder bestimmter Sequenzen (gezielte Resequenzierung) ermöglicht die Identifizierung von genomischen Variationen auf einer breiten Basis. Diese Dissertation beschäftigt sich mit den Herausforderungen, die sich im Zusammenhang mit der Anwendung von NGS-Technologien ergeben. Das beinhaltet die gezielte DNA-Resequenzierung, die Sequenzierung von exprimierten mRNAs (RNA-seq) und microRNAs (miRNA-seq) sowie die Identifizierung von Protein-DNA- Wechselwirkungen, wie Bindungsstellen für Transkriptionsfaktoren oder Histonmodifikationen (ChIP-seq). Die innerhalb der Arbeitsgruppe generierten sowie öffentlich verfügbaren, experimentellen Datensätze wurden verwendet, um neuartige, computergestützte Ansätze und Methoden der Bioinformatik für die Analyse von NGS-Datensätzen zu entwickeln und schließlich biologische Fragen hinsichtlich der Herzfunktion und -krankheit zu beantworten. Eine erste Studie konzentriert sich auf die kombinatorische Regulation von kardialen, DNA- bindenden Transkriptionsfaktoren (ChIP-seq von Srf) beeinflusst von Histonmodifikationen (Histon 3 Acetylierung) und regulatorischen miRNAs (miRNA-seq). Wie in PLoS Genetics im Jahr 2011 veröffentlicht, haben diese verschiedenen regulierenden Ebenen von mRNA-Profilen ein hohes Maß an Wechselwirkung und das Potenzial sich gegenseitig zu modulieren. Zum Beispiel wird die Wirkung von Srf maßgeblich durch das gleichzeitige Auftreten von Histon 3 Acetylierungsmarkierungen beeinflusst. Darüber hinaus können 45% aller differentiell exprimierten mRNAs im Srf Knockdown durch die unterschiedliche Expression von microRNAs erklärt werden. Ungefähr die Hälfte aller differentiell exprimierten mRNAs wird durch andere sekundäre Effekte beeinflusst. Um daher ein vollständiges Bild des regulatorischen Transkriptionsnetzwerkes und der zugrundeliegenden Funktion von Kardiomyozyten (Herzmuskelzellen) zu erhalten, müssen die verschiedenen Modulatoren in Zusammenhang miteinander betrachtet werden. Im Rahmen dieser Studie wurde das Programm MicroRazerS entwickelt (veröffentlicht in Bioinformatics 2010). MicroRazerS ist optimiert für das Mappen kleiner RNA-Sequenzen, wie zum Beispiel microRNAs oder andere kleine nicht-codierende RNAs, zu einem Referenz-Genom. Es zeichnet sich durch eine höhere Sensitivität und zumindest vergleichbare Geschwindigkeit im Vergleich zu anderen Mapping-Programmen aus. Die Ergebnisse zeigen, dass MicroRazerS das Auffinden und die Entdeckung von microRNAs in Hochdurchsatz-Sequenzierungdaten wesentlich erleichtern kann. Ein zweites Projekt zielte darauf ab, die genetische Grundlage der Fallot'schen Tetralogie (TOF) zu identifizieren. TOF tritt in bis zu 10% aller angeborenen Herzerkrankungen auf, die die größte Gruppe der angeborenen Fehlbildungen des Menschen darstellen. Diese Studie zeigt erstmals, dass TOF eine oligogenetische Erkrankung ist. Wir haben eine mehrstufige Studie durchgeführt, darunter die gezielte Resequenzierung von über 1.000 herz- und muskelrelevanten Genen und microRNAs in TOF Patienten, Eltern und Kontrollen sowie die Analyse des ganzen Transkriptoms und miRNomes in TOF Patienten und gesunden Personen unter der Verwendung von NGS-Technologies (87 Proben). Gene wurden nach dem Vorhandensein von schädlichen Variationen und ihrer Mutationsrate in den TOF-Patienten im Vergleich zu gesunden Kontrollen (200 Fälle) beurteilt. Eine Menge von 16 sogenannten TOF-Genen wurde identifiziert, von denen durchschnittlich vier Gene pro TOF-Patient mutiert sind und die die TOF-Patienten von den Kontrollen unterscheiden. Im Allgemeinen stellt die in dieser Studie entwickelte Analysestrategie und der verwendete Bioinformatikansatz eine neue Perspektive für die Analyse von oligo- oder multigenetische Erkrankungen dar

"ZEREPRO" Netzwerk Personalisierte Neurochirurgie: Vortrag gehalten auf dem 9. Medica Tech Forum, 14.11.2018, Düsseldorf

ZEREPRO – Personalisierte Neurochirurgie ist ein Netzwerk, welches seit März 2018 unter der Leitung des Fraunhofer IWU und der Klinik und Poliklinik für Neurochirurgie der Universität als klinischer Partner geführt wird. In dem vom BMWi geförderten Kooperationsnetzwerk arbeiten derzeit 8 Industriepartner und 5 Forschungseinrichtungen an Innovationen im Bereich der Neurochirurgie. Die Vernetzung zwischen Ärzten, Ingenieuren und Medizintechnikunternehmen erlaubt es klinische Frage- und Problemstellungen in eine nutzerfreundliche und vor allem praxistaugliche Lösung zu überführen. Als übergeordnetes Ziel wird die patientenindividuelle Behandlung und Versorgung verfolgt. In einem aus dem Netzwerk entstandenen Projekt werden beispielsweise patientenindividuell gefertigten stereotaktischen Rahmen entwickelt, um hochpräzise neurochirurgische Eingriffe durchzuführen. Die Technologie des 3D-Druckes ermöglicht hierbei die benötigten Geometrien mit entsprechender Genauigkeit zu fertigen und stellt auch die Basis für weitere Projekte, welche sich mit patientenspezifischen Implantaten im Bereich der Neurochirurgie beschäftigen

Medizinische Vorrichtung zum Durchführen einer Biopsie und Verfahren zum Herstellen der medizinischen Vorrichtung

Die vorliegende Erfindung betrifft eine medizinische Vorrichtung zum Durchführen einer Biopsie an einem Bereich (2) eines zu untersuchenden Körpers (8) und ein Verfahren zum Herstellen der medizinischen Vorrichtung. Die medizinische Vorrichtung weist mindestens drei Anschlusselementen (1) zum Anbringen der Vorrichtung an dem Bereich (2) des Körpers (8) oder zum Anbringen der Vorrichtung an Verankerungselemente, die an dem Bereich (2) des Körpers (8) angeordnet sind, eine Hülse (3) als Führung für ein medizinisches Instrument (6), und ein Verbindungselement (4) auf, durch das die mindestens drei Verankerungselemente (1) mit der Hülse (3) verbunden sind. Zumindest das Verbindungselement (4) ist aus einem Leichtbauwerkstoff ausgebildet