Frank Rühle

A genome-wide association study identifies 6p21 as novel risk locus for dilated cardiomyopathy

AIMS Dilated cardiomyopathy (DCM) is one of the leading causes for cardiac transplantations and accounts for up to one-third of all heart failure cases. Since extrinsic and monogenic causes explain only a fraction of all cases, common genetic variants are suspected to contribute to the pathogenesis of DCM, its age of onset, and clinical progression. By a large-scale case-control genome-wide association study we aimed here to identify novel genetic risk loci for DCM. METHODS AND RESULTS Applying a three-staged study design, we analysed more than 4100 DCM cases and 7600 controls. We identified and successfully replicated multiple single nucleotide polymorphism on chromosome 6p21. In the combined analysis, the most significant association signal was obtained for rs9262636 (P = 4.90 × 10(-9)) located in HCG22, which could again be replicated in an independent cohort. Taking advantage of expression quantitative trait loci (eQTL) as molecular phenotypes, we identified rs9262636 as an eQTL for several closely located genes encoding class I and class II major histocompatibility complex heavy chain receptors. CONCLUSION The present study reveals a novel genetic susceptibility locus that clearly underlines the role of genetically driven, inflammatory processes in the pathogenesis of idiopathic DCM.

Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

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Postgwas: Advanced GWAS Interpretation in R

We present a comprehensive toolkit for post-processing, visualization and advanced analysis of GWAS results. In the spirit of comparable tools for gene-expression analysis, we attempt to unify and simplify several procedures that are essential for the interpretation of GWAS results. This includes the generation of advanced Manhattan and regional association plots including rare variant display as well as novel interaction network analysis tools for the investigation of systems-biology aspects. Our package supports virtually all model organisms and represents the first cohesive implementation of such tools for the popular language R. Previous software of that range is dispersed over a wide range of platforms and mostly not adaptable for custom work pipelines. We demonstrate the utility of this package by providing an example workflow on a publicly available dataset.

Long non-coding RNA Databases in Cardiovascular Research

With the rising interest in the regulatory functions of long non-coding RNAs (lncRNAs) in complex human diseases such as cardiovascular diseases, there is an increasing need in public databases offering comprehensive and integrative data for all aspects of these versatile molecules. Recently, a variety of public data repositories that specialized in lncRNAs have been developed, which make use of huge high-throughput data particularly from next-generation sequencing (NGS) approaches. Here, we provide an overview of current lncRNA databases covering basic and functional annotation, lncRNA expression and regulation, interactions with other biomolecules, and genomic variants influencing the structure and function of lncRNAs. The prominent lncRNA antisense noncoding RNA in the INK4 locus (ANRIL), which has been unequivocally associated with coronary artery disease through genome-wide association studies (GWAS), serves as an example to demonstrate the features of each individual database.

Gp130-Dependent Release of Acute Phase Proteins Is Linked to the Activation of Innate Immune Signaling Pathways

Background Elevated levels of acute phase proteins (APP) are often found in patients with cardiovascular diseases. In a previous study, we demonstrated the importance of the IL-6-gp130 axis -as a key regulator of inflammatory acute phase signaling in hepatocytes-for the development of atherosclerosis. Background/Principal Findings Gp130-dependent gene expression was analyzed in a previously established hepatocyte-specific gp130 knockout mouse model. We performed whole transcriptome analysis in isolated hepatocytes to measure tissue specific responses after proinflammatory stimulus with IL-6 across different time points. Our analyses revealed an unexpected small gene cluster that requires IL-6 stimulus for early activation. Several of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Furthermore, we identified a potential biomarker Lipocalin (LCN) 2 for the gp130 dependent early inflammatory response. Conclusions/Significance Our findings suggest a complex network of tightly linked genes involved in the early activation of different parts of the innate immune response including acute phase proteins, complement and coagulation cascade.

Systematic permutation testing in GWAS pathway analyses: identification of genetic networks in dilated cardiomyopathy and ulcerative colitis.

BackgroundGenome wide association studies (GWAS) are applied to identify genetic loci, which are associated with complex traits and human diseases. Analogous to the evolution of gene expression analyses, pathway analyses have emerged as important tools to uncover functional networks of genome-wide association data. Usually, pathway analyses combine statistical methods with a priori available biological knowledge. To determine significance thresholds for associated pathways, correction for multiple testing and over-representation permutation testing is applied.ResultsWe systematically investigated the impact of three different permutation test approaches for over-representation analysis to detect false positive pathway candidates and evaluate them on genome-wide association data of Dilated Cardiomyopathy (DCM) and Ulcerative Colitis (UC). Our results provide evidence that the gold standard - permuting the case–control status – effectively improves specificity of GWAS pathway analysis. Although permutation of SNPs does not maintain linkage disequilibrium (LD), these permutations represent an alternative for GWAS data when case–control permutations are not possible. Gene permutations, however, did not add significantly to the specificity. Finally, we provide estimates on the required number of permutations for the investigated approaches.ConclusionsTo discover potential false positive functional pathway candidates and to support the results from standard statistical tests such as the Hypergeometric test, permutation tests of case control data should be carried out. The most reasonable alternative was case–control permutation, if this is not possible, SNP permutations may be carried out. Our study also demonstrates that significance values converge rapidly with an increasing number of permutations. By applying the described statistical framework we were able to discover axon guidance, focal adhesion and calcium signaling as important DCM-related pathways and Intestinal immune network for IgA production as most significant UC pathway.

Natural genetic variation of the cardiac transcriptome in non-diseased donors and patients with dilated cardiomyopathy

BackgroundGenetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases.ResultsHere we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts.ConclusionsRNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

Rare Variants in the ADAMTS13 Von Willebrand Factor-Binding Domain Contribute to Pediatric Stroke.

Background—Recently, we reported a gene network of ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) genes as central component of the genetic risk contributing to pediatric stroke. ADAMTS13 is a prime example for such a key component as it cleaves von Willebrand factor multimers, reduces platelet adhesion and aggregation, and downregulates thrombus formation and inflammation. Methods and Results—We characterized the genetic architecture of ADAMTS13 through targeted next-generation sequencing of 48 affected children and their unaffected siblings and identified in total 241 variants (single nucleotide polymorphisms or insertions/deletions) in the ADAMTS13 gene. From these, based on significance in the sibship disequilibrium test (P<0.05) or protein-altering properties, we selected 21 common variants covering the complete ADAMTS13 gene for genotyping in 270 trios and subsequent association analyses. Transmission disequilibrium testing was performed for affection status and ADAMTS13 activity levels using PLINK and FBAT, respectively. Ten single nucleotide polymorphisms were significantly associated with pediatric stroke (P<0.05 to P<0.001), 2 of which (rs2285489 and rs28793911) were also significantly associated with ADAMTS13 levels (P=0.0004 and P=0.0092). The resulting protective haplotype H1.1. (T:U 95.5: 144.4; P=0.0016) is associated with increased ADAMTS13 levels (age-adjusted P=0.0108). Haplotype association using a sliding window approach assigns this association to the ADAMTS13 von Willebrand factor–binding domain (P=1.2×10−4). Conclusions—Our data provide a link between the genetic architecture of ADAMTS13, ADAMTS13 levels, and stroke susceptibility. Altogether, these studies render ADAMTS13 an attractive candidate for functional studies and may contribute to personalized diagnosis and treatment options in future.

Paradoxical Effects on Force Generation after Efficient β1-Adrenoceptor Knockdown in Reconstituted Heart Tissue

Stimulation of myocardial β1-adrenoceptors (AR) is a major mechanism that increases cardiac function. We investigated the functional consequences of genetic β1-AR knockdown in three-dimensional engineered heart tissue (EHT). For β1-AR knockdown, short interfering RNA (siRNA) sequences targeting specifically the β1-AR (shB1) and a scrambled control (shCTR) were subcloned into a recombinant adeno-associated virus (AAV)–short hairpin RNA (shRNA) expression system. Transduction efficiency was ∼100%, and radioligand binding revealed 70% lower β1-AR density in AAV6-shB1–transduced EHTs. Force measurements, performed over the culture period of 14 days, showed paradoxically higher force generation in AAV6-shB1 compared with shCTR under basal (0.19 ± 0.01 versus 0.13 ± 0.01 mN) and after β-AR-stimulated conditions with isoprenaline (Δfractional shortening: 72 ± 5% versus 34 ± 4%). Large scale gene expression analysis revealed that AAV6-shCTR compared with nontransduced EHTs showed only few differentially regulated genes (<20), whereas AAV6-shB1 induced marked changes in gene expression (>250 genes), indicating that β1-AR knockdown itself determines the outcome. None of the regulated genes pointed to obvious off-target effects to explain higher force generation. Moreover, compensational regulation of β2-AR signaling or changes in prominent β1-AR downstream targets could be ruled out. In summary, we show paradoxically higher force generation and isoprenaline responses after efficient β1-AR knockdown in EHTs. Our findings 1) reveal an unexpected layer of complexity in gene regulation after specific β1-AR knockdown rather than unspecific dysregulations through transcriptional interference, 2) challenge classic assumptions on the role of cardiac β1-AR, and 3) may open up new avenues for β-AR loss-of-function research in vivo.

Advances in understanding stroke risk in children – a geneticist's view

The advent of the genomic era has provided novel insights into the genetic architecture of common complex diseases, such as thrombophilia and stroke. Since 2006, a growing number of genome wide association studies (GWAS) for common complex diseases have revealed new candidate loci and genomic regions that play an important role in disease aetiology and progression. While GWAS on the above mentioned traits are abundant in adults, similar studies in paediatric study cohorts are lagging behind. However, genetic research in this important clinical area has gained momentum and starts to provide us with exciting insights into the genetic underpinnings of stroke with paediatric onset. Here we review recent advances in genetic association studies underlying stroke in children and aim to translate the results to clinical utility. These studies comprise candidate gene approaches and GWAS, and represent the current status on what we have learnt about the genetic architecture underlying paediatric stroke, and how this may affect medical practice in the years to come.