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PubMed2007

Integrative genomic analysis reveals distinct transcriptional and genetic features associated with chromosome 13 deletion in multiple myeloma.

BACKGROUND AND OBJECTIVES: The chromosome 13 deletion (Delta13) is one of the most frequent chromosomal alterations in multiple myeloma (MM). Delta13 is associated with an unfavorable prognosis, although there is increasing agreement that its prognostic relevance must be related to the ploidy status and the presence of different chromosomal translocations. The aim of this study was to provide a comprehensive analysis of the transcriptional features of Delta13 in MM. DESIGN AND METHODS: Highly purified plasma cells from 80 newly diagnosed MM patients were characterized by means of fluorescence in situ hybridization (FISH) and high-density oligonucleotide microarray for gene expression profiling and chromosomal alterations. RESULTS: We identified 67 differentially expressed genes in the patients with and without the chromosome 13 deletion, all of which were downregulated in the cases with Delta13: 44 mapped along the whole chromosome 13, seven on chromosome 11 and three on chromosome 19. Functional analyses of the selected genes indicated their involvement in protein biosynthesis, ubiquitination and transcriptional regulation. An integrative genomic approach based on regional analyses of the gene expression data identified distinct chromosomal regions whose global expression modulation could differentiate Delta13-positive cases, in particular the upregulation of 1q21-1q42 and the downregulation of 19p and almost the entire chromosome 11. FISH analyses confirmed the close relationship between Delta13-positivity and the presence of extra copies of 1q21-1q42 (p=6 x 10(-4)) or the absence of chromosome 11 and 19 trisomy (p=5 x 10(-4)). INTERPRETATION AND CONCLUSIONS: Our results indicate that distinct types of chromosomal aberrations are closely related to the transcriptional profiles of Delta13-positive cases, suggesting that the contribution of Delta13 to the malignancy should be considered together with associated abnormalities.

Adult↗
PubMed2004

Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy.

We identified three consanguineous Austrian kindreds with 15 members affected by autosomal recessive childhood-onset severe retinal dystrophy, a genetically heterogeneous group of disorders characterized by degeneration of the photoreceptor cells. A whole-genome scan by microarray analysis of single-nucleotide polymorphisms (ref. 2) identified a founder haplotype and defined a critical interval of 1.53 cM on chromosome 14q23.3-q24.1 that contains the gene associated with this form of retinal dystrophy. RDH12 maps in this region and encodes a retinol dehydrogenase proposed to function in the visual cycle. A homozygous 677A-->G transition (resulting in Y226C) in RDH12 was present in all affected family members studied, as well as in two Austrian individuals with sporadic retinal dystrophy. We identified additional mutations in RDH12 in 3 of 89 non-Austrian individuals with retinal dystrophy: a 5-nucleotide deletion (806delCCCTG) and the transition 565C-->T (resulting in Q189X), each in the homozygous state, and 146C-->T (resulting in T49M) and 184C-->T (resulting in R62X) in compound heterozygosity. When expressed in COS-7 cells, Cys226 and Met49 variants had diminished and aberrant activity, respectively, in interconverting isomers of retinol and retinal. The severe visual impairment of individuals with mutations in RDH12 is in marked contrast to the mild visual deficiency in individuals with fundus albipunctatus caused by mutations in RDH5, encoding another retinal dehydrogenase. Our studies show that RDH12 is associated with retinal dystrophy and encodes an enzyme with a unique, nonredundant role in the photoreceptor cells.

Adult↗
PubMed2005

A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes.

The spindle checkpoint prevents chromosome loss by preventing chromosome segregation in cells with improperly attached chromosomes [1, 2 and 3]. The checkpoint senses defects in the attachment of chromosomes to the mitotic spindle [4] and the tension exerted on chromosomes by spindle forces in mitosis [5, 6 and 7]. Because many cancers have defects in chromosome segregation, this checkpoint may be required for survival of tumor cells and may be a target for chemotherapy. We performed a phenotype-based chemical-genetic screen in budding yeast and identified an inhibitor of the spindle checkpoint, called cincreasin. We used a genome-wide collection of yeast gene-deletion strains and traditional genetic and biochemical analysis to show that the target of cincreasin is Mps1, a protein kinase required for checkpoint function [8]. Despite the requirement for Mps1 for sensing both the lack of microtubule attachment and tension at kinetochores, we find concentrations of cincreasin that selectively inhibit the tension-sensitive branch of the spindle checkpoint. At these concentrations, cincreasin causes lethal chromosome missegregation in mutants that display chromosomal instability. Our results demonstrate that Mps1 can be exploited as a target and that inhibiting the tension-sensitive branch of the spindle checkpoint may be a way of selectively killing cancer cells that display chromosomal instability.

Bromobenzenes↗
PubMed2006

Mixed gastric carcinomas show similar chromosomal aberrations in both their diffuse and glandular components.

Gastric cancer is one of the most frequent malignancies in the world. Nonetheless, the knowledge of the molecular events involved in the development of gastric carcinoma is far from complete. One of the hallmarks of gastric cancer is chromosomal instability resulting in abnormal DNA copy number changes throughout the genome. Mixed gastric carcinomas constitute a rare histological entity, containing the two main histological phenotypes (diffuse and intestinal). Very little is known about the underlying mechanisms of phenotypic divergence in these mixed tumours. To the best of our knowledge only E-Cadherin mutations were implicated so far in the divergence of these tumours and nothing is known about the involvement of chromosome copy number changes in the two divergent histological components. In this study, we compared the DNA copy number changes, in the two different components (diffuse and intestinal) of mixed gastric carcinomas by microarray - comparative genomic hybridisation (array CGH). The analysis of 12 mixed gastric carcinomas showed no significant differences in array CGH profiles between the diffuse and intestinal components of mixed carcinomas. This supports the idea that the phenotypic divergence within mixed gastric carcinomas is not caused by DNA chromosomal aberrations.

Chromosome Aberrations↗
PubMed2005

Association of 20q13.2 copy number changes with the advanced stage of ovarian cancer-tissue microarray analysis.

Overrepresentations in 20q have been reported in a number of ovarian cancers by comparative genomic hybridization. In order to study the relation of the increased copy number of 20q13.2 with tumor phenotype in ovarian cancer, we applied FISH on a tissue microarray. The TMA technology enables us to analyze a large number of different malignancy, histology, stage and grade tumors. Overall, the frequency of 20q13.2 alterations in epithelial ovarian cancer was 25.50% (10.74% gains and 14.76% amplifications). There was not statistically significant difference between the frequencies of 20q13.2 copy number changes in different grade tumors. The frequency of gains and amplifications increased significantly from stage I to stage II to stage III tumors. Our results showed strong association between increases 20q13.2 copies and advanced tumor stage. We concluded that genetic alterations in 20q13.2 may be of prognostic significance for stage progression of the ovarian cancer.

Chromosomes, Human, Pair 20↗
PubMed2005

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain.

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.

Brain↗
PubMed2006

Array CGH of fusion gene-positive leukemia-derived cell lines reveals cryptic regions of genomic gain and loss.

Human leukemia-derived cell lines containing characteristic chromosomal translocations and inversions have been instrumental in identifying fusion genes implicated in the pathogenesis of the corresponding leukemia. Although chimeric fusion genes usually provide early and essential steps in the development of leukemia, they are not in themselves sufficient, requiring additional genetic events. The nature of these secondary, cooperating genetic events is not known. The advent of genome wide microarray-based methods for assessing copy number changes made it possible to search for cytogenetically invisible regions of chromosome imbalance. We used BAC microarrays with a resolution of 1 Mb to determine whether cryptic regions of deletion or gain were associated with specific leukemia-associated fusion genes in a series of cell lines. To complement the array analysis, we also applied 24-color karyotyping by M-FISH. This revealed cryptic chromosomal translocations and regions of loss or gain in all the cell lines studied. The chromosomal origin of previously unidentified marker chromosomes was revealed. In all cases, chromosomes described as monosomic were shown to be involved in unbalanced translocations with concurrent loss and/or gain of chromosomal material. The extent of these amplified and deleted regions was more accurately defined. Finally, small regions of deletion and amplification, often including genes known to be involved in leukemia progression (for example MYC, TP53, CDKN2A, and KIT), were identified.

Cell Line, Tumor↗
PubMed2004

High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as probes. The production of BAC/PAC and cDNA arrays is time consuming and expensive. AIM: To evaluate the use of spotted 60 mer oligonucleotides (oligos) for array CGH. METHODS: The hybridisation of tumour cell lines with known chromosomal aberrations on to either BAC or oligoarrrays that are mapped to the human genome. RESULTS: Oligo CGH was able to detect amplifications with high accuracy and greater spatial resolution than other currently used array CGH platforms. In addition, single copy number changes could be detected with a resolution comparable to conventional CGH. CONCLUSIONS: Oligos are easy to handle and flexible, because they can be designed for any part of the genome without the need for laborious amplification procedures. The full genome array, containing around 30000 oligos of all genes in the human genome, will represent a big step forward in the analysis of chromosomal copy number changes. Finally, oligoarray CGH can easily be used for any organism with a fully sequenced genome.

Breast Neoplasms↗
PubMed2005

Prospective gene expression analysis accurately subtypes acute leukaemia in children and establishes a commonality between hyperdiploidy and t(12;21) in acute lymphoblastic leukaemia.

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50-gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6-RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large-scale clinical trials in childhood acute leukaemias.

Acute Disease↗
PubMed2005

A combination of molecular cytogenetic analyses reveals complex genetic alterations in conventional renal cell carcinoma.

Here we report the complex pattern of genomic imbalances and rearrangements in a panel of 19 renal cell carcinoma cell lines detected with molecular cytogenetic analysis. Consistent heterogeneity in chromosome number was found, and most cell lines showed a near-triploid chromosome complement. Several cell lines showed deletions of the TP53 (alias p53), CDKN2A (alias p16), and VHL genes. Multiplex fluorescence in situ hybridization (M-FISH) analysis revealed chromosome 3 translocated to several other partners chromosomes, as well as breakage events commonly affecting chromosomes 1, 5, 8, 10, and 17. The most common abnormality detected with comparative genomic hybridization (CGH) was deletions of chromosome 3p, with loss of the RASSF1, FHIT, and p44S10 loci frequently involved. CGH gain of 5q showed overrepresentation of the EGR1 and CSF1R genes. Recurrent alterations to chromosome 7 included rearrangement of 7q11 and gains of the EGFR, TIF1, and RFC2 genes. Several lines exhibited rearrangement of 12q11 approximately q14 and overrepresentation of CDK4 and SAS loci. M-FISH revealed several other recurrent translocations, and CGH findings included loss of 9p, 14q, and 18q and gain of 8q, 12, and 20. Further genomic microarray changes included loss of MTAP, IGH@, HTR1B, and SMAD4 (previously MADH4) and gains of MYC and TOP1. An excellent correlation was observed between the genomic array and FISH data, demonstrating that this technique is effective and accurate. The aberrations detected here may reflect important pathways in renal cancer pathogenesis.

Carcinoma, Renal Cell↗
PubMed2004

[FISH analysis in the differential diagnosis of flat urothelial lesions using tissue microarrays].

AIMS: FISH technology offers an additional tool in the diagnosis of precancerous and malignant lesions of the urinary tract from cytological specimens. Here, we examined the relevance of chromosomal imbalance in flat urothelial lesions using FISH on paraffin-embedded tissue. In addition, the status of Her2/neu and STK15, a key molecule in the development of aneuploidy, was evaluated. METHODS: Flat lesions (normal urothelium, hyperplasia, reactive atypia, dysplasia and Carcinoma in situlCis) of 73 patients were analyzed in respect of chromosome 3, 7, 17 polysomy, deletion of p16, status of HER2/neu and of STK15 by FISH (UroVysion, PathVysion, Vysis) and immunohistochemistry (HercepTest, DAKO) using tissue microarrays. The data were correlated with histology. RESULTS AND CONCLUSIONS: Aneusomy of at least one of the chromosomes usually correlates with the histology of carcinoma in situ or invasive tumor growth. Reactive atypias, rarely showing chromosomal imbalance, can be distinguished from Cis in the majority of investigated cases, but the FISH technique is not able to differentiate reactive atypia from mild dysplasia. About 30 % of the non-neoplastic lesions like urothelial hyperplasia and normal urothelium display polysomy of at least one chromosome in more than 20% of all cells, indicating an elevated risk towards a synchronous development of a higher dysplastic lesion (i.e. Cis). Polysomy of one of three investigated chromosomes (3, 7, 17) occurs randomly within all lesions. A deletion of the p16 locus is most frequently observed in aneuploid lesions. Altered Her2/neu expression patterns are frequently observed in malignant and dysplastic lesions but also in 25 % of the non-neoplastic lesions. An overexpression of Her2/ neu is found in 10-20% of invasive urothelial carcinomas and occasionally in Cis (5 %). However, the Her2/neu gene locus is not amplified in these samples. Gene amplification of STK15 was seen in tumors as well as in normal urothelium but it is still unclear whether it indicates an elevated risk towards the development of manifest urothelial tumors.

Chromosome Aberrations↗
PubMed2005

Sensitive detection of deletions of one or more exons in the neurofibromatosis type 2 (NF2) gene by multiplexed gene dosage polymerase chain reaction.

Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.

DNA Mutational Analysis↗
PubMed2006

Recurrent cytogenetic aberrations in central neurocytomas and their biological relevance.

Central neurocytomas are rare central nervous system neoplasms. Since the first description, approximately 500 cases of these tumors have been published to date. Nevertheless, only a limited number of genetic studies on these tumors have been reported. Here we investigated 20 "typical" central neurocytomas using array-based comparative genomic hybridization (array-CGH) with the GenoSensor Array 300. The functional significance of detected chromosomal aberrations harboring potent candidate genes was also examined at the mRNA expression level. Each tumor examined displayed DNA copy-number aberrations (CNAs), and mean number of CNAs per tumor was 38.1 +/- 7.1 (range 19-53). Frequent gains were mapped at 2p24.1-22.1, 10q23.3-26.3, 11q23-25, and 18q21.3-qter. Frequent losses were identified at 1pter-36.3, 1p34.3, 6q13-21, 12q23-qter, 17p13.3, 17q11-23, and 20pter-12.3. There were 10 gained and 23 lost single DNA clones affecting >or=40% of samples tested. mRNA expression levels of 24 selected candidate genes harbored in these imbalanced clones were analyzed. MYCN, PTEN, and OR5BF1 were strongly overexpressed, whereas BIN1, SNRPN, and HRAS were found to be strongly underrepresented at the transcriptional level. Thus these data support that MYCN oncogene gain/overexpression accompanied by reduced expression of BIN1 tumor suppressor may contribute to central neurocytoma tumorigenesis.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2005

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana.

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F(2) population obtained from a Col x Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.

Arabidopsis↗
PubMed2006

Micro-array analyses decipher exceptional complex familial chromosomal rearrangement.

Recently there has been an increased interest in large-scale genomic variation and clinically in the consequences of haploinsufficiency of genomic segments or disruption of normal gene function by chromosome rearrangements. Here, we present an extraordinary case in which both mother and daughter presented with unexpected chromosomal rearrangement complexity, which we characterized with array-CGH, array painting and multicolor large insert clone hybridizations. We found the same 12 breakpoints involving four chromosomes in both mother and daughter. In addition, the daughter inherited a microdeletion from her father. We mapped all breakpoints to the resolution level of breakpoint spanning clones. Genes were found within 7 of the 12 breakpoint regions, some of which were disrupted by the chromosome rearrangement. One of the rearrangements disrupted a locus, which has been discussed as a quantitative trait locus for fetal hemoglobin expression in adults. Interestingly, both mother and daughter show persistent fetal hemoglobin levels. We detail the most complicated familial complex chromosomal rearrangement reported to date and thus an extreme example of inheritance of chromosomal rearrangements without error in meiotic segregation.

Child↗
PubMed2005

Microarray-based comparative genomic analyses of the human malaria parasite Plasmodium falciparum using Affymetrix arrays.

Microarray-based comparative genomic hybridization (CGH) provides a powerful tool for whole genome analyses and the rapid detection of genomic variation that underlies virulence and disease. In the field of Plasmodium research, many of the parasite genomes that one might wish to study in a high throughput manner are not laboratory clones, but clinical isolates. One of the key limitations to the use of clinical samples in CGH, however, is the miniscule amounts of genomic DNA available. Here we describe the successful application of multiple displacement amplification (MDA), a non-PCR-based amplification method that exhibits clear advantages over all other currently available methods. Using MDA, CGH was performed on a panel of NF54 and IT/FCR3 clones, identifying previously published deletions on chromosomes 2 and 9 as well as polymorphism in genes associated with disease pathology.

Animals↗
PubMed2007

Escherichia coli with a linear genome.

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.

Cell Proliferation↗
PubMed2003

The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community.

Arabidopsis thaliana is the most widely-studied plant today. The concerted efforts of over 11 000 researchers and 4000 organizations around the world are generating a rich diversity and quantity of information and materials. This information is made available through a comprehensive on-line resource called the Arabidopsis Information Resource (TAIR) (http://arabidopsis.org), which is accessible via commonly used web browsers and can be searched and downloaded in a number of ways. In the last two years, efforts have been focused on increasing data content and diversity, functionally annotating genes and gene products with controlled vocabularies, and improving data retrieval, analysis and visualization tools. New information include sequence polymorphisms including alleles, germplasms and phenotypes, Gene Ontology annotations, gene families, protein information, metabolic pathways, gene expression data from microarray experiments and seed and DNA stocks. New data visualization and analysis tools include SeqViewer, which interactively displays the genome from the whole chromosome down to 10 kb of nucleotide sequence and AraCyc, a metabolic pathway database and map tool that allows overlaying expression data onto the pathway diagrams. Finally, we have recently incorporated seed and DNA stock information from the Arabidopsis Biological Resource Center (ABRC) and implemented a shopping-cart style on-line ordering system.

Arabidopsis↗