Publications by authors named "Suzanne Vanhauwaert"

14 Publications

  • Page 1 of 1

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Genes Chromosomes Cancer 2021 Apr 9;60(4):272-281. Epub 2021 Jan 9.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.
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http://dx.doi.org/10.1002/gcc.22931DOI Listing
April 2021

PHF6 Expression Levels Impact Human Hematopoietic Stem Cell Differentiation.

Front Cell Dev Biol 2020 4;8:599472. Epub 2020 Nov 4.

Department of Diagnostic Sciences, Ghent University, Ghent, Belgium.

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in , encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established models. Our findings reveal that levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of and its downstream target genes. This functional interaction in developing thymocytes was confirmed using a -deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced or notch1 activation. In summary, our work reveals that appropriate control of expression is important for normal human hematopoiesis and provides clues towards the role of in T-ALL development.
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http://dx.doi.org/10.3389/fcell.2020.599472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672048PMC
November 2020

Publisher Correction: In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2019 Jun 4;9(1):8360. Epub 2019 Jun 4.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-44435-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547841PMC
June 2019

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes.

BMC Genomics 2019 Mar 20;20(1):228. Epub 2019 Mar 20.

Department of Biomolecular Medicine & Center for Medical Genetics, Ghent University, 9000, Ghent, Belgium.

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.

Results: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.

Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.
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http://dx.doi.org/10.1186/s12864-019-5608-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425699PMC
March 2019

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PIK-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PIK antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors.

Sci Rep 2018 11 30;8(1):17468. Epub 2018 Nov 30.

Center for Medical Genetics (CMGG), Ghent University, Ghent, Belgium.

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.
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http://dx.doi.org/10.1038/s41598-018-35868-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269481PMC
November 2018

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Nat Commun 2018 11 19;9(1):4866. Epub 2018 Nov 19.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
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http://dx.doi.org/10.1038/s41467-018-06699-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242972PMC
November 2018

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice.

Sci Rep 2018 05 16;8(1):7642. Epub 2018 May 16.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.
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http://dx.doi.org/10.1038/s41598-018-25389-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955877PMC
May 2018

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

Authors:
Jan Van Deun Pieter Mestdagh Patrizia Agostinis Özden Akay Sushma Anand Jasper Anckaert Zoraida Andreu Martinez Tine Baetens Els Beghein Laurence Bertier Geert Berx Janneke Boere Stephanie Boukouris Michel Bremer Dominik Buschmann James B Byrd Clara Casert Lesley Cheng Anna Cmoch Delphine Daveloose Eva De Smedt Seyma Demirsoy Victoria Depoorter Bert Dhondt Tom A P Driedonks Aleksandra Dudek Abdou Elsharawy Ilaria Floris Andrew D Foers Kathrin Gärtner Abhishek D Garg Edward Geeurickx Jan Gettemans Farzaneh Ghazavi Bernd Giebel Tom Groot Kormelink Grace Hancock Hetty Helsmoortel Andrew F Hill Vincent Hyenne Hina Kalra David Kim Joanna Kowal Sandra Kraemer Petra Leidinger Carina Leonelli Yaxuan Liang Lien Lippens Shu Liu Alessandra Lo Cicero Shaun Martin Suresh Mathivanan Prabhu Mathiyalagan Támas Matusek Gloria Milani Marta Monguió-Tortajada Liselot M Mus Dillon C Muth Andrea Németh Esther N M Nolte-'t Hoen Lorraine O'Driscoll Roberta Palmulli Michael W Pfaffl Bjarke Primdal-Bengtson Erminia Romano Quentin Rousseau Susmita Sahoo Natalia Sampaio Monisha Samuel Benjamin Scicluna Bieke Soen Anneleen Steels Johannes V Swinnen Maarit Takatalo Safia Thaminy Clotilde Théry Joeri Tulkens Isabel Van Audenhove Susanne van der Grein Alan Van Goethem Martijn J van Herwijnen Guillaume Van Niel Nadine Van Roy Alexander R Van Vliet Niels Vandamme Suzanne Vanhauwaert Glenn Vergauwen Frederik Verweij Annelynn Wallaert Marca Wauben Kenneth W Witwer Marijke I Zonneveld Olivier De Wever Jo Vandesompele An Hendrix

Nat Methods 2017 02;14(3):228-232

Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.
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http://dx.doi.org/10.1038/nmeth.4185DOI Listing
February 2017

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia.

Haematologica 2014 Dec 24;99(12):1808-16. Epub 2014 Oct 24.

Center for Medical Genetics, Ghent University, Belgium;

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34(+) thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34(+) and Notch inactive CD4(+)CD8(+) thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.
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http://dx.doi.org/10.3324/haematol.2014.115683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258754PMC
December 2014

Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies.

PLoS One 2014 13;9(10):e109091. Epub 2014 Oct 13.

Center of Medical Genetics, Ghent University, Ghent, Belgium.

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109091PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4195698PMC
June 2015

Refinement of the critical 2p25.3 deletion region: the role of MYT1L in intellectual disability and obesity.

Genet Med 2015 Jun 18;17(6):460-6. Epub 2014 Sep 18.

Center for Medical Genetics, Ghent University, Ghent, Belgium.

Purpose: Submicroscopic deletions of chromosome band 2p25.3 are associated with intellectual disability and/or central obesity. Although MYT1L is believed to be a critical gene responsible for intellectual disability, so far no unequivocal data have confirmed this hypothesis.

Methods: In this study we evaluated a cohort of 22 patients (15 sporadic patients and two families) with a 2p25.3 aberration to further refine the clinical phenotype and to delineate the role of MYT1L in intellectual disability and obesity. In addition, myt1l spatiotemporal expression in zebrafish embryos was analyzed by quantitative polymerase chain reaction and whole-mount in situ hybridization.

Results: Complete MYT1L deletion, intragenic deletion, or duplication was observed in all sporadic patients, in addition to two patients with a de novo point mutation in MYT1L. The familial cases comprise a 6-Mb deletion in a father and his three children and a 5' MYT1L overlapping duplication in a father and his two children. Expression analysis in zebrafish embryos shows specific myt1l expression in the developing brain.

Conclusion: Our data strongly strengthen the hypothesis that MYT1L is the causal gene for the observed syndromal intellectual disability. Moreover, because 17 patients present with obesity/overweight, haploinsufficiency of MYT1L might predispose to weight problems with childhood onset.Genet Med 17 6, 460-466.
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http://dx.doi.org/10.1038/gim.2014.124DOI Listing
June 2015

Defective initiation of glycosaminoglycan synthesis due to B3GALT6 mutations causes a pleiotropic Ehlers-Danlos-syndrome-like connective tissue disorder.

Am J Hum Genet 2013 Jun 9;92(6):935-45. Epub 2013 May 9.

Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium. Electronic address:

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs(∗)163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals' fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.
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http://dx.doi.org/10.1016/j.ajhg.2013.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675258PMC
June 2013