Showing papers by "Jo Vandesompele published in 2007"

qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data

Abstract: Although quantitative PCR (qPCR) is becoming the method of choice for expression profiling of selected genes, accurate and straightforward processing of the raw measurements remains a major hurdle. Here we outline advanced and universally applicable models for relative quantification and inter-run calibration with proper error propagation along the entire calculation track. These models and algorithms are implemented in qBase, a free program for the management and automated analysis of qPCR data.

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

Abstract: 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.

Molecular dissection of isolated disease features in mosaic neurofibromatosis type 1.

Abstract: Elucidation of the biological framework underlying the development of neurofibromatosis type 1 (NF1)–related symptoms has proved to be difficult. Complicating factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types, and the NF1-haploinsufficient state of all cells in the body. Here, we investigate three patients with distinct NF1-associated clinical manifestations (neurofibromas only, pigmentary changes only, and association of both symptoms). For each patient, various tissues and cell types were tested with comprehensive and quantitative assays capable of detecting low-percentage NF1 mutations. This approach confirmed the biallelic NF1 inactivation in Schwann cells in neurofibromas and, for the first time, demonstrated biallelic NF1 inactivation in melanocytes in NF1-related cafe-au-lait macules. Interestingly, both disease features arise even within a background of predominantly NF1 wild-type cells. Together, the data provide molecular evidence that (1) the distinct clinical picture of the patients is due to mosaicism for the NF1 mutation and (2) the mosaic phenotype reflects the embryonic timing and, accordingly, the neural crest–derived cell type involved in the somatic NF1 mutation. The study of the affected cell types provides important insight into developmental concepts underlying particular NF1-related disease features and opens avenues for improved diagnosis and genetic counseling of individuals with mosaic NF1.

ArrayCGH-based classification of neuroblastoma into genomic subgroups

Abstract: High-resolution array comparative genomic hybridization (arrayCGH) profiling was performed on 75 primary tumors and 29 cell lines to gain further insight into the genetic heterogeneity of neuroblastoma and to refine genomic subclassification. Using a novel data-mining strategy, three major and two minor genomic subclasses were delineated. Eighty-three percent of tumors could be assigned to the three major genomic subclasses, corresponding to the three known clinically and biologically relevant subsets in neuroblastoma. The remaining subclasses represented (1) tumors with no/few copy number alterations or an atypical pattern of aberrations and (2) tumors with 11q13 amplification. Inspection of individual arrayCGH profiles showed that recurrent genomic imbalances were not exclusively associated with a specific subclass. Of particular notice were tumors with numerical imbalances typically observed in subtype 1 neuroblastoma, in association with genomic features of subtype 2A or 2B. A search for prognostically relevant genomic alterations disclosed 1q gain as a predictive marker for therapy failure within the group of subtype 2A and 2B tumors. In cell lines, a high incidence of 6q loss was observed, with a 3.87-5.32 Mb region of common loss within 6q25.1-6q25.2. Our study clearly illustrates the importance of genomic profiling in relation to tumor behavior in neuroblastoma. We propose that genome-wide assessment of copy number alterations should ideally be included in the genetic workup of neuroblastoma. Further multicentric studies on large tumor series are warranted in order to improve therapeutic stratification in conjunction with other features such as age at diagnosis, tumor stage, and gene expression signatures.

Real-time quantitative PCR as an alternative to Southern blot or fluorescence in situ hybridization for detection of gene copy number changes.

Abstract: Changes in copy number of genes contribute to the pathogenesis of various genetic disorders and cancer. The status of a gene has not only diagnostic value but sometimes directs treatment stratification. Although, for many years, Southern blot and fluorescence in situ hybridization were the standard methods for the detection of deletion, duplication, or amplification of a gene, both methods have their own important limitations. Recently, realtime quantitative PCR has proven to be a good alternative for the detection of gene copy number changes. Its main advantages are the large dynamic range of accurate quantification, the absence of post-PCR manipulations, its high-throughput screening capacity and degree of automation, and the possibility to perform the assay on minimal amounts of sample DNA in just a few hours of time. In this chapter, we outline the procedure of how to develop an assay for the detection of gene copy number changes for your gene of interest. We illustrate the approach by describing a validated assay for the detection of germline VHL exon deletions and for determination of MYCN copy numbers in tumor samples.

Erratum: Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes (Genome Biology)

Abstract: A correction to Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes by K De Preter, J Vandesompele, P Heimann, N Yigit, S Beckman, A Schramm, A Eggert, RL Stallings, Y Benoit, M Renard, A De Paepe, G Laureys, S Påhlman and F Speleman. Genome Biology 2006 7:R84

Identification and expression analysis of genes associated with bovine blastocyst formation

Abstract: Background Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation. First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures.

Translating expression profiling into a clinically feasible test to predict neuroblastoma outcome.

Abstract: Purpose: To assess the feasibility of predicting neuroblastoma outcome using highly parallel quantitative real-time PCR data. Experimental Design: We generated expression profiles of 63 neuroblastoma patients, 47 of which were analyzed by both Affymetrix U95A microarrays and highly parallel real-time PCR on microfluidic cards (MFC; Applied Biosystems). Top-ranked genes discriminating patients with event-free survival or relapse according to high-level analysis of Affymetrix chip data, as well as known neuroblastoma marker genes ( MYCN and NTRK1/TrkA ), were quantified simultaneously by real-time PCR. Analysis of PCR data was accomplished using high-level bioinformatics methods including prediction analysis of microarray, significance analysis of microarray, and Computerized Affected Sibling Pair Analyzer and Reporter. Results: Internal validation of the MFC method proved it highly reproducible. Correlation of MFC and chip expression data varied markedly for some genes. Outcome prediction using prediction analysis of microarray on real-time PCR data resulted in 80% accuracy, which is comparable to results obtained using the Affymetrix platform. Real-time PCR data were useful for risk assessment of relapsing neuroblastoma ( P = 0.0006, log-rank test) when Computerized Affected Sibling Pair Analyzer and Reporter analysis was applied. Conclusions: These data suggest that multiplex real-time PCR might be a promising approach to reduce the complexity of information obtained from whole-genome array experiments. It could provide a more convenient and less expensive tool for routine application in a clinical setting.

Identification of 2 putative critical segments of 17q gain in neuroblastoma through integrative genomics

Abstract: Partial gain of chromosome arm 17q is the most frequent genetic change in neuroblastoma (NB) and constitutes the strongest independent genetic factor for adverse prognosis. It is assumed that 1 or more genes on 17q contribute to NB pathogenesis by a gene dosage effect. In the present study, we applied chromosome 17 tiling path BAC arrays on a panel of 69 primary tumors and 28 NB cell lines in order to reduce the current smallest region of gain and facilitate identification of candidate dosage sensitive genes. In all tumors and cell lines with 17q gain, large distal segments were consistently present in extra copies and no interstitial gains were observed. In addition to these large regions of distal gain with breakpoints proximal to coordinate 44.3 Mb (17q21.32), smaller regions of gain (distal to coordinate 60 Mb at 17q24.1) were found superimposed on the larger region in a minority of cases. Positional gene enrichment analysis for 17q genes overexpressed in NB showed that dosage sensitive NB oncogenes are most likely located in the gained region immediately distal to the most distal breakpoint of the 2 breakpoint regions. Interestingly, comparison of gene expression profiles between primary tumors and normal fetal adrenal neuroblasts revealed 2 gene clusters on chromosome 17q that are overexpressed in NB, i.e. a region on 17q21.32 immediately distal to the most distal breakpoint (in cases with single regions of gain) and 17q24.1, a region coinciding with breakpoints leading to superimposed gain.

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

Abstract: 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.

Rapid and easy prenatal diagnosis of sickle cell anemia using double-dye LNA probe technology.

Abstract: Taking advantage of the high specificity of double-dye hydrolysis-locked nucleic acid (LNA) probes (Ugozzoli et al, 2004), we developed a qualitative three-colour real-time polymerase chain reaction (PCR) assay for the genotyping of sickle cell disease (SCD). The term SCD encompasses a group of disorders associated with mutations in the Haemoglobin Beta gene (HBB). The most common forms are defined by the presence, in codon 6, of an E6V mutation (GAG > GTG; Haemoglobin S; Hb S) on both alleles (sickle cell anaemia) and co-inheritance of Hb S and Hb C (E6K mutation; GAG > AAG; Hb SC disease). The highest prevalence of SCD is found in subSaharan Africa (sickle-cell gene carrier prevalence varying between 5% and 40% of the population), and most affected children die in the early years of life without being diagnosed or because of non-adapted treatments (Weatherall & Clegg, 2001). The methodological approach presented here combined the use of a primer pair designed to amplify the region of interest in the HBB gene and three double-dye probes containing LNA nucleotides. Each probe was labelled with a different fluorochrome to enable the different alleles to be discriminated (Fig 1). Primers were designed with the freeware meltcalc, ver. 2.0 (http://www.meltcalc.de). The Tm of each double-dye LNA probe was calculated by using the Exiqon Tm prediction tool (http://lna-tm.com). Samples from patients that were homozygous for each of the three aforementioned alleles were used to demonstrate the specificity of the probes (Fig 1). We used unique PCR conditions on the ABI 7500 Fast machine (Applied Biosystems, Foster City, CA, USA), which generated specific results for each probe in <40 min (see Table SI, Fig S1). A cohort of 37 genomic DNA representing the different possible genotypes was used to compare our new technique with either a PCR followed by restriction endonuclease cleavage to detect the A and S alleles (Saiki et al, 1988), or an allele-specific PCR designed to detect A and C alleles (Fischel-Ghodsian et al, 1990). A 100% concordance was observed between our technology and the two published methods. Moreover, the deduced genotypes matched perfectly with the available phenotypic analysis (Gulbis et al, 2006). To investigate the sensitivity of this analysis, we tested different amounts of genomic DNA per PCR reaction. The results were perfectly interpretable from 100 to 1 ng of starting material (data not shown), and enabled the testing of prenatal diagnosis samples even when there was scarce fetal tissue (demonstrated on 18 gDNA obtained from fetal samples; data not shown). Finally, to demonstrate the robustness of our approach, we tested 10 representative samples on currently available instru-