PubMed Health⌕ Search

Biomedical subjects

Jo Vandesompele

Publications and source records attributed to Jo Vandesompele.

At least 19 recordsLinked to original sources
PubMed2007

High resolution tiling-path BAC array deletion mapping suggests commonly involved 3p21-p22 tumor suppressor genes in neuroblastoma and more frequent tumors.

The recurrent loss of 3p segments in neuroblastoma suggests the implication of 1 or more tumor suppressor genes but thus far few efforts have been made to pinpoint their detailed chromosomal position. To achieve this goal, array-based comparative genomic hybridization was performed on a panel of 23 neuroblastoma cell lines and 75 primary tumors using a tiling-path bacterial artificial chromosome array for chromosome 3p. A total of 45 chromosome 3 losses were detected, including whole chromosome losses, large terminal deletions and interstitial deletions. The latter, observed in cell lines as well as a number of distal deletions detected in primary tumors, allowed us to demarcate 3 minimal regions of loss of 3.6 Mb [3p21.31-p21.2, shortest regions of overlap (SRO)1], 1.4 Mb (3p22.3-3p22.2, SRO2) and 3.8 Mb (3p25.3-p25.1, SRO3) in size. The present data significantly extend previous findings and now firmly establish critical regions on 3p implicated in neuroblastoma. Interestingly, the 2 proximal regions coincide with previously defined SROs on 3p21.3 in more frequent tumors including lung and breast cancer. As such, similar tumor suppressor genes may play a critical role in development or progression of a variety of neoplasms, including neuroblastoma.

Cell Line, Tumor↗
PubMed2006

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis.

BACKGROUND: DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers. RESULTS: To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast. CONCLUSION: We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.

Algorithms↗
PubMed2006

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A.

BACKGROUND: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor gamma coactivator 1alpha (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. RESULTS: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). CONCLUSION: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

Adipose Tissue↗
PubMed2006

Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma.

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

Apoptosis↗
PubMed2006

The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma.

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.

Blotting, Western↗
PubMed2006

RTPrimerDB: the real-time PCR primer and probe database, major update 2006.

The RTPrimerDB (http://medgen.ugent.be/rtprimerdb) project provides a freely accessible data retrieval system and an in silico assay evaluation pipeline for real-time quantitative PCR assays. Over the last year the number of user submitted assays has grown to 3500. Data conveyance from Entrez Gene by establishing an assay-to-gene relationship enables the addition of new primer assays for one of the 1.5 million different genes from 2300 species stored in the system. Easy access to the primer and probe data is possible by using multiple search criteria. Assay reports contain gene information, assay details (such as oligonucleotide sequences, detection chemistry and reaction conditions), publication information, users' experimental evaluation feedback and submitter's contact details. Gene expression assays are extended with a scalable assay viewer that provides detailed information on the alignment of primer and probe sequences on the known transcript variants of a gene, along with Single Nucleotide Polymorphisms (SNP) positions and peptide domain information. Furthermore, an mfold module is implemented to predict the secondary structure of the amplicon sequence, as this has been reported to impact the efficiency of the PCR. RTPrimerDB is also extended with an in silico analysis pipeline to streamline the evaluation of custom designed primer and probe sequences prior to ordering and experimental evaluation. In a secured environment, the pipeline performs automated BLAST specificity searches, mfold secondary structure prediction, SNP or plain sequence error identification, and graphical visualization of the aligned primer and probe sequences on the target gene.

Computer Graphics↗
PubMed2006

Translocation-excision-deletion-amplification mechanism leading to nonsyntenic coamplification of MYC and ATBF1.

Despite oncogene amplification being a characteristic of many tumor types, the mechanisms leading to amplicon formation have remained largely unresolved. In this study, we used a combinatorial approach of fluorescence in situ hybridization and single-nucleotide polymorphism chip gene copy number analyses to unravel the mechanism leading to nonsyntenic coamplification of MYC and ATBF1 in SJNB-12 cells. To explain our findings, we propose a complex series of events consisting of multiple double-strand breaks, accompanied (or triggered) by the formation of a reciprocal translocation t(8;16), as well as excisions and deletions near the translocation breakpoints. This study provides evidence for a translocation-excision-deletion-amplification sequence of events rather than a breakage-fusion-bridge model, which has been more frequently proposed to explain proto-oncogene amplification. Furthermore, it illustrates the power of presently available tools for detailed analysis of the complex rearrangements that accompany amplicon formation.

Cell Line, Tumor↗
PubMed2006

EVI1 is consistently expressed as principal transcript in common and rare recurrent 3q26 rearrangements.

In contrast to the well-documented involvement of EVI1 in various 3q26 aberrations, the transcriptional status of EVI1 in rare recurrent or sporadic 3q26 chromosomal defects has remained largely unexplored. Moreover, in a recent report, the association between 3q26 alterations in myeloid proliferations and ectopic EVI1 expression was questioned. Therefore, we performed a detailed physical mapping of 3q26 breakpoints using a 1.3-Mb tiling path BAC contig covering the EVI1 locus and a carefully designed quantification of both EVI1 and MDS/EVI1 transcripts in 30 hematological malignancies displaying 3q26 aberrations. Cases included well-known rare, recurring chromosomal aberrations such as t(3;17)(q26;q22), t(2;3)(p21-22;q26), and t(3;6)(q26;q25), as well as 10 new sporadic cases. Extensive 3q26 breakpoint mapping allowed unequivocal and sensitive FISH detection of EVI1 rearrangements on both metaphases and interphase nuclei. Real-time quantitative PCR analyses indicated that typically both MDS1/EVI1 and EVI1, but not MDS1, were expressed in these malignancies, with EVI1 the primary transcript. In conclusion, we have demonstrated EVI1 involvement in numerous novel sporadic and recurrent 3q26 rearrangements. Our results underscore the feasibility of FISH as an adjunct to PCR for the identification of EVI1 deranged leukemias and identified EVI1 as the principal transcript expressed in these malignancies.

Adolescent↗
PubMed2006

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas.

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes.

BACKGROUND: Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands. RESULTS: Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis. CONCLUSION: This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.

Databases, Genetic↗
PubMed2005

Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells.

BACKGROUND: Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable. METHODS: We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay. RESULTS: The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 10(6) mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 10(4) mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value. CONCLUSIONS: ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.

Adolescent↗
PubMed2005

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.

BACKGROUND: Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the successful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. RESULTS: In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. CONCLUSION: Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

Animals↗
PubMed2005

Expression profiling suggests underexpression of the GABA(A) receptor subunit delta in the fragile X knockout mouse model.

It is still unclear why absence of the fragile X protein (FMRP) leads to mental retardation and specific behavioral problems. In neurons, the protein transports specific mRNAs towards the actively translating ribosomes near the synapses. To unravel the mechanism leading to the disorder, we performed global gene expression analysis by means of the differential display method using the fragile X mouse model. To verify differential expression, we used microarray technology and real-time PCR. Three differentially expressed cDNAs showed consistent underexpression in the fragile X knockout mouse, including a GABA(A) receptor subunit delta, a Rho guanine exchange factor 12 and an EST BU563433. In addition, we identified 5 genes that showed differential expression dependent on the sample of RNA analysis. We consider their differential expression as provisional. It is possible that these differentially expressed genes play an important role in the cognitive and behavioral problems observed in the fragile X syndrome.

Animals↗
PubMed2005

Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11.

BACKGROUND: Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11. RESULTS: In a first step, we performed high-resolution array CGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13-->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene. CONCLUSION: Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.

Alleles↗
PubMed2005

arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays.

BACKGROUND: The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. RESULTS: We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. CONCLUSION: ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at http://medgen.ugent.be/arrayCGHbase/.

Base Sequence↗
PubMed2005

Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma.

PURPOSE: Neuroblastoma is a genetically heterogeneous pediatric tumor with a remarkably variable clinical behavior ranging from widely disseminated disease to spontaneous regression. In this study, we aimed for comprehensive genetic subgroup discovery and assessment of independent prognostic markers based on genome-wide aberrations detected by comparative genomic hybridization (CGH). MATERIALS AND METHODS: Published CGH data from 231 primary untreated neuroblastomas were converted to a digitized format suitable for global data mining, subgroup discovery, and multivariate survival analyses. RESULTS: In contrast to previous reports, which included only a few genetic parameters, we present here for the first time a strategy that allows unbiased evaluation of all genetic imbalances detected by CGH. The presented approach firmly established the existence of three different clinicogenetic subgroups and indicated that chromosome 17 status and tumor stage were the only independent significant predictors for patient outcome. Important new findings were: (1) a normal chromosome 17 status as a delineator of a subgroup of presumed favorable-stage tumors with highly increased risk; (2) the recognition of a survivor signature conferring 100% 5-year survival for stage 1, 2, and 4S tumors presenting with whole chromosome 17 gain; and (3) the identification of 3p deletion as a hallmark of older age at diagnosis. CONCLUSION: We propose a new regression model for improved patient outcome prediction, incorporating tumor stage, chromosome 17, and amplification/deletion status. These findings may prove highly valuable with respect to more reliable risk assessment, evaluation of clinical results, and optimization of current treatment protocols.

Adolescent↗