Systematic evaluation of the effect of common SNPs onpre-mRNA splicing

Background: The evolutionary and biomedical importance of differential mRNA splicing is well established, especially with regard to pathophysiological conditions. Up to 60% of mutations that contribute to disease development have been proposed to do so by disrupting splicing events. Erroneous splice site usage is also observed in numerous diseases. Problem: Identification and functional annotation of single-nucleotides polymorphisms that interfere with splicing mechanisms (‘splice SNPs') is a major challenge and needs to be supported by an efficient method. Solution: 1) A high-throughput methodology was established to facilitate the screening of allele-dependent splicing in a high-throughput fashion (ElSharawy et al., 2006). The method integrated a package of four new software tools and was mainly based on using a panel of 92 matched pairs of individual-specific gDNA and cDNA samples. For each SNP, 16 cDNAs providing a balanced representation of the genotypes at the respective SNP were investigated by nested RT-PCR and subsequent sequencing. Putative allele-dependent splicing events were verified by cloning and sequencing. 2) A systematic, SNP-centered approach was followed and the database dbSNP was screened to filter a group of common SNPs at either canonical splice sites or ESEs that were classified as putatively splicing-relevant by bioinformatics tools. This was completed in two screening rounds using web-based tools (Alex’s splice site score calculator and ESEfinder) and neural network, respectively. A group of SNPs at NAGNAG tandem repeat sites was also tested (ElSharawy et al., 2008). Results and conclusion: As a result of genotyping, the 223 non-redundant candidate SNPs were experimentally tested, and 18 allele-dependent splicing events were identified, of which 15 were novel and 3 exhibited an already known functional relevance. However, the positive predictive value of the bioinformatics tools turned out to be low, ranging from 0% for ESEFinder to 9% (in the case of acceptor site SNPs) for the neural network. Overall, the currently available bioinformatics tools contribute little to the understanding as to how common genetic variation impacts mRNA splicing. Therefore, there is a need for an alternative system. A proof of concept and outlook: The present study made some preliminary steps to develop a novel in vitro fluorescence-based splice reporter system. The ongoing systematic and hypothesis-driven experiments, which combine the advantages of FACS-based reporter constructs with a dichromatic readout method (a defined and experimentally controlled system) and ultra-high-throughput second generation sequencing technology, will serve to establish an efficient means to address many splice-related topics, and thus, would improve our understanding of mammalian splice site anatomy.Hintergrund: Die weitreichende evolutionäre und physiologische Bedeutung des differentiellen mRNA-Spleißens ist allgemein bekannt, besonders im Hinblick auf pathophysiologische und biomedizinische Fragestellungen. Man geht davon aus, dass bis zu 60 Prozent aller krankheitsverursachenden Mutationen auf eine Zerstörung von Spleißstellen zurückzuführen sind. Eine fehlerhafte Nutzung von vorhandenen Spleißstellen ist bereits für eine Vielzahl von Krankheiten bekannt. Problemstellung: Die Identifizierung und funktionelle Annotation spleißrelevanter SNPs stellt eine große Herausforderung dar und bedarf der Unterstützung durch eine effiziente Methodik. Lösungsansatz: 1)Zur Erleichterung des Screenings nach allelabhängigen Spleißereignissen wurde eine neue Hochdurchsatzmethodik entwickelt (ElSharawy et al., 2006). Diese umfasst vier neue Software-Anwendungen und basiert hauptsächlich auf der Nutzung eines Panels von 92 übereinstimmenden Paaren individuenspezifischer gDNA- und cDNA-Proben. Für jeden der zu untersuchenden SNPs wurden 16 cDNAs mittels RT-PCR und anschließender Sequenzierung untersucht. Allelabhängige Spleißereignisse wurden durch Klonierung und Sequenzierung verifiziert. 2)In einem systematischen, SNP-zentrierten Ansatz wurden häufige SNPs an kanonischen Spleißstellen sowie an ESEs aus dbSNP gefiltert und mittels webbasierter Anwendungen als potentiell spleißrelevant klassifiziert. In einem zweiten Ansatz erfolgte die Klassifizierung der SNPs mittels eines neuronalen Netzwerkes. Die als spleißrelevant klassifizierten SNPs wurden im Anschluß mit der oben beschriebenen Methode untersucht. Zusätzlich wurde eine Gruppe von SNPs an NAGNAG Tandems Repeats getestet (ElSharawy et al., 2008). Ergebnisse und Schlussfolgerungen: Insgesamt wurden 223 nicht redundante Kandidaten-SNPs experimentell getestet. Dabei wurden 18 allelabhängige Spleißvorgänge identifiziert, von denen 15 neuartig waren und für 3 die funktionelle Relevanz bekannt ist. Dabei stellte sich die korrekte positive Vorhersagefähigkeit der bioinformatischen Tools als äußerst gering heraus - von 9% (für Spleißakzeptor-SNPs) für das neuronale Netzwerk bis zu 0% für den „ESEFinder“. Zusammenfassend konnten die verwendeten bioinformatischen Anwendungen nur wenig zum Verständnis beitragen, wie häufige genetische Variationen das mRNA-Spleißen beeinflussen. Ausblick: In der vorliegenden Arbeit wurden entscheidende vorläufige Schritte zur Entwicklung eines neuartigen fluoreszenzbasierten in vitro-Spleißreportersystems geleistet, welches zur Zeit getestet und im Hinblick auf gezielte Fragestellungen validiert wird. Die momentan durchgeführten systematischen und hypothesenorientierten Experimente kombinieren die Vorteile FACS-basierter dichromatischer Reportersyteme mit denen der Hochdurchsatz-Sequenziertechnologie (second generation sequencing technology) und könnten ein effizientes Mittel zur Aufklärung vieler spleißrelevanter Fragestellungen darstellen und unser Verständnis des allelabhängigen Spleißens entscheidend verbessern

hsa‐miR‐374b‐5p regulates expression of the gene U2AF homology motif (UHM) kinase 1

Objective: We aimed to identify a microRNA (miRNA) that is significantly upregulated in blood and in cells of the oral mucosa upon exposure to the periodontitis main risk factors oral inflammation and tobacco smoke, to subsequently identify its target gene and to describe the molecular mechanism of gene regulation. Background: miRNAs are associated with many disorders. Array-based miRNA expression studies indicated a number of differentially expressed miRNAs in the pathology of oral diseases. However, these miRNAs mostly lacked replication, and their target genes have remained unknown. Methods: 863 miRNAs were analyzed in blood from 18 PD cases and 70 controls (Geniom Biochip). Selected miRNAs were analyzed for upregulation in the inflamed oral mucosa of PD patients using published miRNA expression profiling studies from gingival cells. hsa-miR-374b-5p mimic was overexpressed in primary gingival fibroblasts (pGFs) from 3 donors, and genome-wide mRNA expression was quantified (Clarion Array). Gene-specific regulation was validated by qRT-PCR and Luciferase activity in HeLa cells. Results: hsa-miR-374b-5p showed >twofold change (FC) in 3 independent studies performed in blood, gingival tissues, and cells. After hsa-miR-374b-5p overexpression, genome-wide expression analysis showed UHMK1 as top 1 downregulated gene in pGFs (p = 2.5 × 10-04 , fold change = -1.8). Reporter genes demonstrated that hsa-miR-374b-5p downregulates mRNA levels (p = .02; FC = -1.5), leading to reduction in protein activity (p = .013, FC = -1.3). Conclusions: hsa-miR-374b-5p is upregulated in blood and ginvial cells exposed to oral inflammation and tobacco smoke and regulates UHMK1, which has a role in osteoclast differentiation

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached

From Next-Generation Sequencing Alignments to Accurate Comparison and Validation of Single-Nucleotide Variants: The Pibase Software

Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5–60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences

Systematic Association Mapping Identifies NELL1 as a Novel IBD Disease Gene

Crohn disease (CD), a sub-entity of inflammatory bowel disease (IBD), is a complex polygenic disorder. Although recent studies have successfully identified CD-associated genetic variants, these susceptibility loci explain only a fraction of the heritability of the disease. Here, we report on a multi-stage genome-wide scan of 393 German CD cases and 399 controls. Among the 116,161 single-nucleotide polymorphisms tested, an association with the known CD susceptibility gene NOD2, the 5q31 haplotype, and the recently reported CD locus at 5p13.1 was confirmed. In addition, SNP rs1793004 in the gene encoding nel-like 1 precursor (NELL1, chromosome 11p15.1) showed a consistent disease-association in independent German population- and family-based samples (942 cases, 1082 controls, 375 trios). Subsequent fine mapping and replication in an independent sample of 454 French/Canadian CD trios supported the authenticity of the NELL1 association. Further confirmation in a large German ulcerative colitis (UC) sample indicated that NELL1 is a ubiquitous IBD susceptibility locus (combined p<10−6; OR = 1.66, 95% CI: 1.30–2.11). The novel 5p13.1 locus was also replicated in the French/Canadian sample and in an independent UK CD patient panel (453 cases, 521 controls, combined p<10−6 for SNP rs1992660). Several associations were replicated in at least one independent sample, point to an involvement of ITGB6 (upstream), GRM8 (downstream), OR5V1 (downstream), PPP3R2 (downstream), NM_152575 (upstream) and HNF4G (intron)

Mutational Characterization of the Bile Acid Receptor TGR5 in Primary Sclerosing Cholangitis

TGR5, the G protein-coupled bile acid receptor 1 (GPBAR1), has been linked to inflammatory pathways as well as bile homeostasis, and could therefore be involved in primary sclerosing cholangitis (PSC) a chronic inflammatory bile duct disease. We aimed to extensively investigate TGR5 sequence variation in PSC, as well as functionally characterize detected variants. Complete resequencing of TGR5 was performed in 267 PSC patients and 274 healthy controls. Six nonsynonymous mutations were identified in addition to 16 other novel single-nucleotide polymorphisms. To investigate the impact from the nonsynonymous variants on TGR5, we created a receptor model, and introduced mutated TGR5 constructs into human epithelial cell lines. By using confocal microscopy, flow cytometry and a cAMP-sensitive luciferase assay, five of the nonsynonymous mutations (W83R, V178M, A217P, S272G and Q296X) were found to reduce or abolish TGR5 function. Fine-mapping of the previously reported PSC and UC associated locus at chromosome 2q35 in large patient panels revealed an overall association between the TGR5 single-nucleotide polymorphism rs11554825 and PSC (odds ratio = 1.14, 95% confidence interval: 1.03-1.26, p = 0.010) and UC (odds ratio = 1.19, 95% confidence interval 1.11-1.27, p = 8.5 x 10(-7)), but strong linkage disequilibrium precluded demarcation of TGR5 from neighboring genes. Resequencing of TGR5 along with functional investigations of novel variants provided unique insight into an important candidate gene for several inflammatory and metabolic conditions. While significant TGR5 associations were detected in both UC and PSC, further studies are needed to conclusively define the role of TGR5 variation in these diseases

Plasmonic Metaparticles on a Blackbody Create Vivid Reflective Colors for Naked-Eye Environmental and Clinical Biodetection

Plasmonic dipoles are famous for their strong absorptivity rather than their reflectivity. Here, the as‐yet unknown specular reflection and the Brewster effect of ultrafine plasmonic dipoles, metaparticles, are introduced and exploited as the basis of new design rules for advanced applications. A configuration of “Plasmonic metaparticles on a blackbody” is demonstrated and utilized for the design of a tailored perfect‐colored absorber and for visual detection of environmental dielectrics that is not readily done by extinction plasmonics. Moreover, the Plasmonic Brewster Wavelength (PBW) effect is introduced as a new platform for the naked‐eye and bulk biodetection of analytes. The technique operates based on slight changes of molecular polarizability which is not detectable via conventional plasmon resonance techniques. As a specific highlight, the clinical applicability of the PBW method is demonstrated while addressing the transduction plasmonic techniques' challenge in detection of bulk refractive index changes of the healthy and diseased human serum exosomes. Finally, the sputtering‐based fabrication method used here is simple, inexpensive, and scalable, and does not require the sophisticated patterning approach of lithography or precise alignment of light coupling for the biodetection

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing

Abstract Background Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). Results We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with Conclusions We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.</p

Targeted enrichment of genomic DNA regions for next-generation sequencing

In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings

miRNAs can be generally associated with human pathologies as exemplified for miR-144*

Background: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. Methods: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. Results: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 ( 95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. Conclusions: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers