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Frank Speleman

Publications and source records attributed to Frank Speleman.

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↗
PubMed2007

Osteopoikilosis, short stature and mental retardation as key features of a new microdeletion syndrome on 12q14.

This report presents the detection of a heterozygous deletion at chromosome 12q14 in three unrelated patients with a similar phenotype consisting of mild mental retardation, failure to thrive in infancy, proportionate short stature and osteopoikilosis as the most characteristic features. In each case, this interstitial deletion was found using molecular karyotyping. The deletion occurred as a de novo event and varied between 3.44 and 6 megabases (Mb) in size with a 3.44 Mb common deleted region. The deleted interval was not flanked by low-copy repeats or segmental duplications. It contains 13 RefSeq genes, including LEMD3, which was previously shown to be the causal gene for osteopoikilosis. The observation of osteopoikilosis lesions should facilitate recognition of this new microdeletion syndrome among children with failure to thrive, short stature and learning disabilities.

Abnormalities, Multiple↗
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

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

Molecular pathogenesis of multiple gastrointestinal stromal tumors in NF1 patients.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. KIT and PDGFRA activating mutations are the oncogenic mechanisms in most sporadic GISTs. In addition to sporadic occurrences, GISTs are increasingly being recognized in association with neurofibromatosis type 1 (NF1), yet the underlying pathogenic mechanism remains elusive. To gain an insight into the mechanisms underlying GIST formation in NF1 patients, we studied seven GISTs from three NF1 patients with a combination of different techniques: mutation analysis (KIT, PDGFRA and NF1), western blotting, array CGH and ex vivo imatinib response experiments. We demonstrate that (i) the NF1-related GISTs do not have KIT or PDGFRA mutations, (ii) the molecular event underlying GIST development in this patient group is a somatic inactivation of the wild-type NF1 allele in the tumor and (iii) inactivation of neurofibromin is an alternate mechanism to (hyper) activate the MAP-kinase pathway, while the JAK-STAT3 and PI3K-AKT pathways are less activated in NF1-related GIST compared with sporadic GISTs. In conclusion, we report for the first time the molecular pathogenesis of GISTs in NF1 individuals and demonstrate that this type of tumor clearly belongs to the spectrum of clinical symptoms in NF1.

Adult↗
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

GAB2 is a novel target of 11q amplification in AML/MDS.

Chromosome arm 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are rare but recurrent aberrations in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We have recently shown that in addition to the MLL core amplicon, independent sequences in 11q23-24 and/or 11q13.5 are coamplified within the same cytogenetic markers in 90% and 60% of patients, respectively. Here we further narrow down the minimal amplicon in 11q13.5 to 1.17 Mb by means of semi-quantitative PCR and FISH analyses. The newly defined amplicon contains seven genes, including the GRB2-associated binding protein 2 (GAB2). Using real-time RT-PCR we show a significant transcriptional upregulation of GAB2 in the patients who have GAB2 coamplified with MLL. Thus, the adaptor molecule GAB2 that has already been shown to enhance oncogenic signaling in other neoplasias appears as a novel target of 11q amplification in AML/MDS.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Somatic loss of wild type NF1 allele in neurofibromas: Comparison of NF1 microdeletion and non-microdeletion patients.

Neurofibromatosis type I (NF1) is an autosomal dominant familial tumor syndrome characterized by the presence of multiple benign neurofibromas. In 95% of NF1 individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the germline mutation consists of a microdeletion that includes the NF1 gene and several flanking genes. We studied the frequency of loss of heterozygosity (LOH) in the NF1 region as a mechanism of somatic NF1 inactivation in neurofibromas from NF1 patients with and without a microdeletion. There was a statistically significant difference between these two patient groups in the proportion of neurofibromas with LOH. None of the 40 neurofibromas from six different NF1 microdeletion patients showed LOH, whereas LOH was observed in 6/28 neurofibromas from five patients with an intragenic NF1 mutation (P = 0.0034, Fisher's exact). LOH of the NF1 microdeletion region in NF1 microdeletion patients would de facto lead to a nullizygous state of the genes located in the deletion region and might be lethal. The mechanisms leading to LOH were further analyzed in six neurofibromas. In two out of six neurofibromas, a chromosomal microdeletion was found; in three, a mitotic recombination was responsible for the observed LOH; and in one, a chromosome loss with reduplication was present. These data show an important difference in the mechanisms of second hit formation in the 2 NF1 patient groups. We conclude that NF1 is a familial tumor syndrome in which the type of germline mutation influences the type of second hit in the tumors.

Alleles↗
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

HOXA gene cluster rearrangement in a t(7;9)(p15;q34) in a child with MDS.

We describe the molecular characterization of a t(7;9)(p15;q34) found in a 15-month-old female patient, diagnosed with refractory anemia with excess blasts in transformation (RAEBt), in progression to acute myeloid leukemia (AML) M7. Molecular characterization of the 7p15 breakpoint showed that this was localized within a fully sequenced PAC clone RP1-170O19 containing the HOXA4 to HOXA13 genes and the EVX1 gene. The 9q34 breakpoint was mapped distal to ABL1 and proximal to NOTCH1 excluding their involvement as fusion gene partners. Our findings suggest a causal role for HOXA genes in childhood myelodysplasia and warrant investigation of this locus in a larger series of patients.

Chromosomes, Human, Pair 7↗
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

A novel gene family NBPF: intricate structure generated by gene duplications during primate evolution.

Partial and complete genome duplications occurred during evolution and resulted in the creation of new genes and gene families. We identified a novel and intricate human gene family located primarily in regions of segmental duplications on human chromosome 1. We named it NBPF, for neuroblastoma breakpoint family, because one of its members is disrupted by a chromosomal translocation in a neuroblastoma patient. The NBPF genes have a repetitive structure with high intragenic and intergenic sequence similarity in both coding and noncoding regions. These similarities might expose these genomic regions to illegitimate recombination, resulting in structural variation in the NBPF genes. The encoded proteins contain a highly conserved domain of unknown function, which we have named the NBPF repeat. In silico analysis combined with the isolation of multiple full-length cDNA clones showed that several members of this gene family are abundantly expressed in a large variety of tissues and cell lines. Strikingly, no discernable orthologues could be identified in the completed genomes of fruit fly, nematode, mouse, or rat, but sequences with low homology could be isolated from the draft canine and bovine genomes. Interestingly, this gene family shows primate-specific duplications that result in species-specific arrays of NBPF homologous sequences. Overall, this novel NBPF family reflects the continuous evolution of primate genomes that resulted in large physiological differences, and its potential role in this process is discussed.

Amino Acid Sequence↗
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↗