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Congenital heart defects (CHDs) are the most common birth defect in human with an incidence of almost 1% of all live births. Most cases have a multifactorial origin with both genetics and the environment playing a role in its development and progression. Adding an epigenetic component to this aspect is exemplified by monozygotic twins which share the same genetic background but have a different disease status. As a result, the interplay between the genetic, epigenetic and the environmental conditions might contribute to the etiology and phenotype. To date, the underlying causes of the majority of CHDs remain poorly understood. In this study, we performed genome-wide high-throughput sequencing to examine the genetic, structural genomic and epigenetic differences of two identical twin pairs discordant for Tetralogy of Fallot (TOF), representing the most common cyanotic form of CHDs. Our results show the almost identical genetic and structural genomic identity of the twins. In contrast, several epigenetic alterations could be observed given by DNA methylation changes in regulatory regions of known cardiac-relevant genes. Overall, this study provides first insights into the impact of genetic and especially epigenetic factors underlying monozygotic twins discordant for CHD like TOF.

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.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.

Background/Aim: Anthracyclines have been proven able to reduce the activity of vinca alkaloids by induction of cell-cycle arrest. The present study aims at identifying the critical initiation steps of signal transduction which transduce the inhibitory effects on the cytotoxicity of vinca alkaloids. MATERIALS AND METHODS: Several new cytostatic drug classes were evaluated together with vincristine in tumor cell lines and patients' tumor cells. RNA interference was used for molecular analyses. RESULTS: Inhibition of vincristine was observed by all cytostatic drugs, which induced cell-cycle arrest. Knockdown of proteins of the DNA damage response ascribed the inhibitory effect to a common pathway involving Chk-1, p53 and p21. Upstream of Chk-1 signal transduction depended on both ATM and ATR for all drugs except methotrexate. CONCLUSION: We have identified critical signaling steps of the DNA damage response system activated by cytostatic drugs, which reduce the anti-tumor activity of vinca alkaloids. The obtained results encourage the development of novel therapeutic strategies to prevent pathway interactions based on the molecular understanding of drug action and drug-drug interactions. &nbsp

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.