[Show abstract][Hide abstract] ABSTRACT: Background: Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM-stages II-III. Prognostic biomarkers for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. MicroRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR based relative quantification of miRNA expression is lacking. Methods: A selection of frozen tumor specimens from two independent patient cohorts with TNM-stage II-III microsatellite stable primary adenocarcinomas were used for laser capture microdissection. Next generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N=50). Differential expression analysis, comparing in metastasized- and non-metastasized tumors, identified prognostic microRNAs. Validation was performed on colon tumors from the German cohort (N=43) using qPCR. Results: MiR-25-3p and miR-339-5p were identified and validated as independent prognostic markers and used to construct a multivariate nomogram for metastasis risk prediction. The nomogram showed good probability prediction in validation. Additionally, we recommend combination of miR-16-5p and miR-26a-5p as standard for normalization in qPCR of colon cancer tissue-derived microRNA expression. Conclusions: In this international study we identified and validated a miRNA classifier in primary cancers, and propose a nomogram capable of predicting metastasis risk in microsatellite stable TNM-stage II-III colon cancer. Impact: In conjunction with TNM-staging, by means of a nomogram, this microRNA classifier may allow for personalized treatment decisions based on individual tumor characteristics.
[Show abstract][Hide abstract] ABSTRACT: Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1-EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects.European Journal of Human Genetics advance online publication, 16 July 2014; doi:10.1038/ejhg.2014.132.
European journal of human genetics: EJHG 07/2014; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fieldsof biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 07/2014; · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labor-intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the 6 independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.
Experimental Cell Research 05/2014; · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive, or uncharacterized regions.
We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by TALENs and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics.
We have implemented TSSV as a Python-package that can be installed through the command-line using pip install tssv command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.
[Show abstract][Hide abstract] ABSTRACT: Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project-the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
[Show abstract][Hide abstract] ABSTRACT: RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and g
[Show abstract][Hide abstract] ABSTRACT: RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and guidelines for assessing technical biases in RNA-seq data.
[Show abstract][Hide abstract] ABSTRACT: Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA-mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.
Nucleic Acids Research 06/2013; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin - a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (http://www.lovd.nl/MTM1), with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2-15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1-5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.European Journal of Human Genetics advance online publication, 12 September 2012; doi:10.1038/ejhg.2012.201.
European journal of human genetics: EJHG 09/2012; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used array analysis to establish the presence of microRNAs and mRNA in cell-derived vesicles from many sources. Here, we used an unbiased approach by deep sequencing of small RNA released by immune cells. We found a large variety of small non-coding RNA species representing pervasive transcripts or RNA cleavage products overlapping with protein coding regions, repeat sequences or structural RNAs. Many of these RNAs were enriched relative to cellular RNA, indicating that cells destine specific RNAs for extracellular release. Among the most abundant small RNAs in shuttle RNA were sequences derived from vault RNA, Y-RNA and specific tRNAs. Many of the highly abundant small non-coding transcripts in shuttle RNA are evolutionary well-conserved and have previously been associated to gene regulatory functions. These findings allude to a wider range of biological effects that could be mediated by shuttle RNA than previously expected. Moreover, the data present leads for unraveling how cells modify the function of other cells via transfer of specific non-coding RNA species.
Nucleic Acids Research 07/2012; 40(18):9272-85. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n = 35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1-150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.
PLoS ONE 06/2012; 7(6):e38298. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key step in heart development is the coor-dinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that elec-trically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coor-dination of these important processes in heart development.
Cellular and Molecular Life Sciences CMLS 04/2012; 69(8):1377-1389. · 5.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Treatment of disorders of the sinus node or the atrioventricular node requires insights into the molecular mechanisms of development and homoeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogramme terminally differentiated working cardiomyocytes into pacemaker cells.
TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice using tamoxifen. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na(+) channel (Na(V)1.5; I(Na)), and inwardly rectifying K(+) ion channels (K(ir) genes; I(K1)). Concordantly, we observed conduction slowing in these hearts and reductions in I(Na) and I(K1) in cardiomyocytes isolated from these hearts. The reduction in I(K1) resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current I(f) was observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes.
TBX3 reprogrammes terminally differentiated working cardiomyocytes and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce I(K1) density to enable diastolic depolarization are promising TBX3 characteristics that may facilitate biological pacemaker formation strategies.
Cardiovascular Research 03/2012; 94(3):439-49. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key step in heart development is the coordinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that electrically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coordination of these important processes in heart development.
Cellular and Molecular Life Sciences CMLS 12/2011; 69(8):1377-89. · 5.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis.
PLoS ONE 12/2010; 5(12):e15504. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are small non-coding RNA transcripts that regulate post-transcriptional gene expression. The millions of short sequence reads generated by next generation sequencing technologies make this technique explicitly suitable for profiling of known and novel microRNAs. A modification to the small-RNA expression kit (SREK, Ambion) library preparation method for the SOLiD sequencing platform is described to generate microRNA sequencing libraries that are compatible with the Illumina Genome Analyzer.
High quality sequencing libraries can successfully be prepared from as little as 100 ng small RNA enriched RNA. An easy to use perl-based analysis pipeline called E-miR was developed to handle the sequencing data in several automated steps including data format conversion, 3' adapter removal, genome alignment and annotation to non-coding RNA transcripts. The sample preparation and E-miR pipeline were used to identify 37 cardiac enriched microRNAs in stage 16 chicken embryos. Isomir expression profiles between the heart and embryo were highly correlated for all miRNAs suggesting that tissue or cell specific miRNA modifications do not occur.
In conclusion, our alternative sample preparation method can successfully be applied to generate high quality miRNA sequencing libraries for the Illumina genome analyzer.
[Show abstract][Hide abstract] ABSTRACT: The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium-specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node-specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.
Circulation Research 07/2009; 105(1):61-9. · 11.09 Impact Factor