GPFrontend and GPGraphics: graphical analysis tools for genetic association studies

<p>Abstract</p> <p>Background</p> <p>Most software packages for whole genome association studies are non-graphical, purely text based programs originally designed to run with UNIX-like operating systems. Graphical output is often not intended or supposed to be performed with other command line tools, e.g. gnuplot.</p> <p>Results</p> <p>Using the Microsoft .NET 2.0 platform and Visual Studio 2005, we have created a graphical software package to analyze data from microarray whole genome association studies, both for a DNA-pooling based approach as well as regular single sample data. Part of this package was made to integrate with GenePool 0.8.2, a previously existing software suite for GNU/Linux systems, which we have modified to run in a Microsoft Windows environment. Further modifications cause it to generate some additional data. This enables GenePool to interact with the .NET parts created by us. The programs we developed are GPFrontend, a graphical user interface and frontend to use GenePool and create metadata files for it, and GPGraphics, a program to further analyze and graphically evaluate output of different WGA analysis programs, among them also GenePool.</p> <p>Conclusions</p> <p>Our programs enable regular MS Windows users without much experience in bioinformatics to easily visualize whole genome data from a variety of sources.</p

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells

Tracing Myelin Protein Zero (P0) in vivo by construction of P0-GFP fusion proteins

BACKGROUND: Mutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein. RESULTS: In contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed. CONCLUSIONS: The present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

Although defects in intestinal barrier function are discussed as a key pathogenic factor in patients with inflammatory bowel diseases (IBD), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we performed a novel approach to characterize the transcriptome of IECs from IBD patients using a genome wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBD where Rho-A activation was suppressed due to reduced expression of the Rho-A prenylation enzyme GGTase-I. The functional relevance of this pathway was highlighted by studies in mice with conditional gene targeting in which deletion of RhoA or GGTase-I in IECs caused spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4+ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBD. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I deficient IECs, our findings open new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients

Dissecting TSC2-mutated renal and hepatic angiomyolipomas in an individual with ARID1B-associated intellectual disability

Background Several subunits of the SWI/SNF chromatin remodeling complex are implicated in both cancer and neurodevelopmental disorders (NDD). Though there is no clinical evidence for an increased tumor risk in individuals with NDDs due to germline mutations in most of these genes so far, this has been repeatedly proposed and discussed. A young woman with NDD due to a de novo mutation in ARID1B now presented with a large renal (> 19 cm in diameter) and multiple hepatic angiomyolipomas (AMLs) but no other signs of tuberous sclerosis complex. Methods We analyzed tumor and healthy tissue samples with exome and panel sequencing. Results Additionally to the previously known, germline ARID1B variant we identified a post-zygotic truncating TSC2 variant in both renal and hepatic AMLs but not in any of the healthy tissues. We did not detect any further, obvious tumor driver events. The identification of a passenger variant in SIPA1L3 in both AMLs points to a common clonal origin. Metastasis of the renal AML into the liver is unlikely on the basis of discordant histopathological features. Our findings therefore point to very low-grade mosaicism for the TSC2 variant, possibly in a yet unknown mesenchymal precursor cell that expanded clonally during tumor development. A possible contribution of the germline ARID1B variant to the tumorigenesis remains unclear but cannot be excluded given the absence of any other evident tumor drivers in the AMLs. Conclusion This unique case highlights the blurred line between tumor genetics and post-zygotic events that can complicate exact molecular diagnoses in patients with rare manifestations. It also demonstrates the relevance of multiple disorders in a single individual, the challenges of detecting low-grade mosaicisms, and the importance of proper diagnosis for treatment and surveillance

Genetics of serum urate concentrations and gout in a high risk population, patients with chronic kidney disease

We evaluated genetics of hyperuricemia and gout, their interaction with kidney function and medication intake in chronic kidney disease (CKD) patients. Genome-wide association studies (GWAS) of urate and gout were performed in 4941 CKD patients in the German Chronic Kidney Disease (GCKD) study. Effect estimates of 26 known urate-associated population-based single nucleotide polymorphisms (SNPs) were examined. Interactions of urate-associated variants with urate-altering medications and clinical characteristics of gout were evaluated. Genome-wide significant associations with serum urate and gout were identified for known loci at SLC2A9 and ABCG2, but not for novel loci. Effects of the 26 known SNPs were of similar magnitude in CKD patients compared to population based individuals, except for SNPs at ABCG2 that showed greater effects in CKD. Gene-medication interactions were not significant when accounting for multiple testing. Associations with gout in specific joints were significant for SLC2A9 rs12498742 in wrists and midfoot joints. Known genetic variants in SLC2A9 and ABCG2 were associated with urate and gout in a CKD cohort, with effect sizes for ABCG2 significantly greater in CKD compared to the general population. CKD patients are at high risk of gout due to reduced kidney function, diuretics intake and genetic predisposition, making treatment to target challenging

Reset of inflammatory priming of joint tissue and reduction of the severity of arthritis flares by bromodomain inhibition

OBJECTIVE: We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extra-terminal motif (BET) proteins translate epigenetic changes into transcription. Here we used a BET inhibitor to target inflammatory tissue priming and reduce flare severity in experimental arthritis. METHODS: BALB/c mice were treated intraperitoneally or locally into the paw with I-BET151, which blocks interaction of BET proteins with acetylated histones. Effect of I-BET151 on acute arthritis and/or inflammatory tissue priming was assessed in a model of repeated injections of monosodium urate crystals or zymosan into the paw. I-BET151 was given either from before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq) and functional analysis (invasion, cytokine production, migration, senescence, metabolic flux) was performed on murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS: Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic and pathogenic phenotype of SFs. CONCLUSION: Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, which makes them promising therapeutic targets for preventing arthritis flares in previously affected joints

Expanding the clinical spectrum associated with defects in CNTNAP2 and NRXN1

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Heterozygous copy-number and missense variants in CNTNAP2 and NRXN1 have repeatedly been associated with a wide spectrum of neuropsychiatric disorders such as developmental language and autism spectrum disorders, epilepsy and schizophrenia. Recently, homozygous or compound heterozygous defects in either gene were reported as causative for severe intellectual disability. Methods 99 patients with severe intellectual disability and resemblance to Pitt-Hopkins syndrome and/or suspected recessive inheritance were screened for mutations in CNTNAP2 and NRXN1. Molecular karyotyping was performed in 45 patients. In 8 further patients with variable intellectual disability and heterozygous deletions in either CNTNAP2 or NRXN1, the remaining allele was sequenced. Results By molecular karyotyping and mutational screening of CNTNAP2 and NRXN1 in a group of severely intellectually disabled patients we identified a heterozygous deletion in NRXN1 in one patient and heterozygous splice-site, frameshift and stop mutations in CNTNAP2 in four patients, respectively. Neither in these patients nor in eight further patients with heterozygous deletions within NRXN1 or CNTNAP2 we could identify a defect on the second allele. One deletion in NRXN1 and one deletion in CNTNAP2 occurred de novo, in another family the deletion was also identified in the mother who had learning difficulties, and in all other tested families one parent was shown to be healthy carrier of the respective deletion or mutation. Conclusions We report on patients with heterozygous defects in CNTNAP2 or NRXN1 associated with severe intellectual disability, which has only been reported for recessive defects before. These results expand the spectrum of phenotypic severity in patients with heterozygous defects in either gene. The large variability between severely affected patients and mildly affected or asymptomatic carrier parents might suggest the presence of a second hit, not necessarily located in the same gene.Peer Reviewe

Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis

Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking—but not forced alcohol exposure—reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-016-1658-6) contains supplementary material, which is available to authorized users