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PubMed2006

Identification of overexpressed genes in frequently gained/amplified chromosome regions in multiple myeloma.

BACKGROUND AND OBJECTIVES: Multiple myeloma (MM) is a malignancy characterized by clonal expansion of plasma cells. In 50% of the cases, the neoplastic transformation begins with a chromosomal translocation that juxtaposes the IGH gene locus to an oncogene. Gene copy number changes are also frequent in MM but less characterized than in other neoplasias. We aimed to characterize genes that are amplified and overexpressed in human myeloma cell lines (HMCL) to provide putative molecular targets for MM therapy. DESIGN AND METHODS: Nine HMCL were characterized by fluorescent in situ hybridization, comparative genomic hybridization (CGH) and cDNA microarrays for gene expression profiling and copy number changes. RESULTS: After defining the IGH-translocations present in the cell lines, we conducted expression-profiling analysis. Supervised analysis identified 166 genes with significantly different expression among the cell lines harboring MMSET/FGFR3 (4p16), MAF (16q) and CCND1 (11q13) rearrangements. Array-CGH was then performed. Five chromosomes recurrently affected by gains/amplifications in primary samples and cell lines were analyzed in detail. Sixty amplified and overexpressed genes were found and 25 (42%) of them were only overexpressed when amplified; moreover, six showed a significant association between overexpression and gain/amplification. We also found co-amplification and overexpression for genes located within the same amplicons, such as MALT1 and BCL2. INTERPRETATION AND CONCLUSIONS: Parallel analysis of gene copy numbers and expression levels by cDNA microarray in MM allowed efficient identification of genes whose expression levels are elevated because of increased copy number. This is the first time that MALT1 and BCL2 have been shown to be overexpressed and amplified in MM.

Caspases↗
PubMed2006

MES-4: an autosome-associated histone methyltransferase that participates in silencing the X chromosomes in the C. elegans germ line.

Germ cell development in C. elegans requires that the X chromosomes be globally silenced during mitosis and early meiosis. We previously found that the nuclear proteins MES-2, MES-3, MES-4 and MES-6 regulate the different chromatin states of autosomes versus X chromosomes and are required for germline viability. Strikingly, the SET-domain protein MES-4 is concentrated on autosomes and excluded from the X chromosomes. Here, we show that MES-4 has histone H3 methyltransferase (HMT) activity in vitro, and is required for histone H3K36 dimethylation in mitotic and early meiotic germline nuclei and early embryos. MES-4 appears unlinked to transcription elongation, thus distinguishing it from other known H3K36 HMTs. Based on microarray analysis, loss of MES-4 leads to derepression of X-linked genes in the germ line. We discuss how an autosomally associated HMT may participate in silencing genes on the X chromosome, in coordination with the direct silencing effects of the other MES proteins.

Animals↗
PubMed2004

Accurate detection of aneuploidies in array CGH and gene expression microarray data.

MOTIVATION: Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data. RESULTS: We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells. AVAILABILITY: Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/

Algorithms↗
PubMed2004

Genetic analysis of genome-wide variation in human gene expression.

Natural variation in gene expression is extensive in humans and other organisms, and variation in the baseline expression level of many genes has a heritable component. To localize the genetic determinants of these quantitative traits (expression phenotypes) in humans, we used microarrays to measure gene expression levels and performed genome-wide linkage analysis for expression levels of 3,554 genes in 14 large families. For approximately 1,000 expression phenotypes, there was significant evidence of linkage to specific chromosomal regions. Both cis- and trans-acting loci regulate variation in the expression levels of genes, although most act in trans. Many gene expression phenotypes are influenced by several genetic determinants. Furthermore, we found hotspots of transcriptional regulation where significant evidence of linkage for several expression phenotypes (up to 31) coincides, and expression levels of many genes that share the same regulatory region are significantly correlated. The combination of microarray techniques for phenotyping and linkage analysis for quantitative traits allows the genetic mapping of determinants that contribute to variation in human gene expression.

Alleles↗
PubMed2004

Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination.

Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

Chromosomes↗
PubMed2006

GenRate: a generative model that reveals novel transcripts in genome-tiling microarray data.

Genome-wide microarray designs containing millions to hundreds of millions of probes are available for a variety of mammals, including mouse and human. These genome tiling arrays can potentially lead to significant advances in science and medicine, e.g., by indicating new genes and alternative primary and secondary transcripts. While bottom-up pattern matching techniques (e.g., hierarchical clustering) can be used to find gene structures in microarray data, we believe the many interacting hidden variables and complex noise patterns more naturally lead to an analysis based on generative models. We describe a generative model of tiling data and show how the sum-product algorithm can be used to infer hybridization noise, probe sensitivity, new transcripts, and alternative transcripts. The method, called GenRate, maximizes a global scoring function that enables multiple transcripts to compete for ownership of putative probes. We apply GenRate to a new exon tiling dataset from mouse chromosome 4 and show that it makes significantly more predictions than a previously described hierarchical clustering method at the same false positive rate. GenRate correctly predicts many known genes and also predicts new gene structures. As new problems arise, additional hidden variables can be incorporated into the model in a principled fashion, so we believe that GenRate will prove to be a useful tool in the new era of genome-wide tiling microarray analysis.

Animals↗
PubMed2003

Genetic susceptibility to nickel-induced acute lung injury.

Human exposure to insoluble and soluble nickel compounds is extensive. Besides wide usage in many industries, nickel compounds are contained in cigarette smoke and, in low levels, in ambient particulate matter. Soluble nickel particulate, especially nickel sulfate (NiSO(4)), has been associated with acute lung injury. To begin identifying genes controlling susceptibility to NiSO(4), mean survival times (MSTs) of eight inbred mouse strains were determined after aerosol exposure. Whereas A/J (A) mice were sensitive, C57BL/6J (B6) mice survived nearly twice as long (resistant). Their offspring were similarly resistant, demonstrating heritability as a dominant trait. Quantitative trait locus (QTL) analysis of backcross mice generated from these strains identified a region on chromosome 6 significantly linked to survival time. Regions on chromosomes 1 and 12 were suggestive of linkage and regions on chromosomes 8, 9, and 16 contributed to the response. Haplotype analysis demonstrated that QTLs on chromosomes 6, 9, 12, and 16 could explain the MST difference between the parental strains. To complement QTL analysis results, cDNA microarray analysis was assessed following NiSO(4) exposure of A and B6 mice. Significant expression changes were identified in one or both strains for >100 known genes. Closer evaluation of these changes revealed a temporal pattern of increased cell proliferation, extracellular matrix repair, hypoxia, and oxidative stress, followed by diminished surfactant proteins. Certain expressed sequence tags clustered with known genes, suggesting possible co-regulation and novel roles in pulmonary injury. Together, results from QTL and microarray analyses of nickel-induced acute lung injury survival allowed us to generate a short list of candidate genes.

Adaptation, Physiological↗
PubMed2004

Microarray analysis of transposition targets in Escherichia coli: the impact of transcription.

Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.

Bacteriophage mu↗
PubMed2004

A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.

Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

Animals↗
PubMed2005

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

Animals↗
PubMed2005

A microarray analysis of developmentally regulated genes during macronuclear differentiation in the stichotrichous ciliate Stylonychia lemnae.

After sexual reproduction in ciliated protozoa a new macronucleus differentiates from a micronuclear derivative. In the course of macronuclear development dramatic DNA- and chromatin reorganisation processes occur, which include splicing of DNA sequences such as IES (internal eliminated sequences) and transposon-like elements during formation of polytene chromosomes, degradation of the polytene chromosomes and specific elimination of micronuclear-specific DNA, de novo addition of telomeres and specific amplification of DNA sequences. In order to understand the molecular basis of this nuclear differentiation process, analysis of developmentally regulated genes seems to be a necessary prerequisite. We performed a microarray analysis to identify genes differentially expressed during macronuclear differentiation. 467 sequences from cDNA libraries were identified as possible candidates from which 384 sequences were further characterised by sequence analysis. These sequences were identified, if possible, by DNA and protein BLAST analysis. Expression of one of these sequences was silenced by RNAi and a preliminary functional analysis performed. Results presented in this study provide the basis for a functional characterisation of genes differentially expressed during this nuclear differentiation process.

Animals↗
PubMed2005

An Xq22.3 duplication detected by comparative genomic hybridization microarray (Array-CGH) defines a new locus (FGS5) for FG syndrome.

FG syndrome is an X-linked multiple congenital anomalies (MCA) syndrome. It has been mapped to four distinct loci FGS1-4, through linkage analysis (Xq13, Xp22.3, and Xp11.4-p11.3) and based on the breakpoints of an X chromosome inversion (Xq11:Xq28), but so far no gene has been identified. We describe a boy with FG syndrome who has an inherited duplication at band Xq22.3 detected by comparative genomic hybridization microarray (Array-CGH). These duplication maps outside all four loci described so far for FG syndrome, representing therefore a new locus, which we propose to be called FGS5. MID2, a gene closely related to MID1, which is known to be mutated in Opitz G/BBB syndrome, maps within the duplicated segment of our patient. Since FG and Opitz G/BBB syndromes share many manifestations we considered MID2 a candidate gene for FG syndrome. We also discuss the involvement of other potential genes within the duplicated segment and its relationship with clinical symptoms of our patient, as well as the laboratory abnormalities found in his mother, a carrier of the duplication.

Abnormalities, Multiple↗
PubMed2006

Mapping the distribution of chromatin proteins by ChIP on chip.

The ChIP on chip method combines chromatin immunoprecipitation (ChIP) with hybridization on DNA microarrays (chip). The ChIP technique allows one to obtain a DNA sample enriched in sequences bound by transcription factors or chromatin-associated proteins. Usually, ChIP is used to test whether specific candidate sequences are bound by a transcription factor, but microarrays are a powerful tool that allows testing large pools of sequences at once. This chapter presents the pipeline of a ChIP on chip method that can be applied to map the binding sites of chromatin-associated proteins along Drosophila chromosomes at different developmental stages. This chapter provides protocols for ChIP, for quality control tests of ChIP samples, for microarray design, for hybridization of the ChIP samples onto microarrays, and for initial analysis of the data. In addition, this chapter discusses the most important steps in each of the protocols as well as the importance of bioinformatic analysis in order to extract valuable biological information from the data sets.

Chromatin↗
PubMed2003

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1.

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

Adolescent↗
PubMed2006

Global gene expression of a murein (Braun) lipoprotein mutant of Salmonella enterica serovar Typhimurium by microarray analysis.

Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.

Flagella↗
PubMed2007

Deletion mapping in Xp21 for patients with complex glycerol kinase deficiency using SNP mapping arrays.

Infantile or complex glycerol kinase deficiency (cGKD) is a contiguous gene deletion syndrome caused by a loss of GK (MIM# 300474), along with its neighboring genes, Duchenne muscular dystrophy (DMD; MIM# 300377) and/or Nuclear Receptor Subfamily 0, Group B, Member 1 (NR0B1; MIM# 300473). Patients with cGKD present with glyceroluria and hyperglycerolemia in association with DMD and/or adrenal hypoplasia congenita (AHC). The purpose of these investigations was to determine whether the Affymetrix GeneChip Mapping Array (SNP chip) could be utilized to detect and map breakpoints in patients with cGKD. Genomic DNAs from several primary lymphoblastoid cell lines from patients with cGKD were analyzed on the Affymetrix platform. The Affymetrix SNP chip is a high-density oligonucleotide array that allows a standardized, parallel interrogation of thousands of SNPs across the entire genome (except for the Y chromosome). Analysis of the array features' hybridization intensities enabled clear delineation of the patient deletions with a high degree of confidence. Many of these patient deletions had been mapped by PCR and their breakpoints confirmed by sequencing. This study demonstrates the utility of the Affymetrix Mapping GeneChips for molecular cytogenetic analysis, beyond the SNP genotyping for which the arrays were initially designed. With one out of 160 live births (approximately 25,000 U.S. neonates annually) reported to have cytogenetic disorders, we envision a significant need for such a standardized platform to carry out rapid, high-throughput, genomic analyses for molecular cytogenetics applications.

Cells, Cultured↗
PubMed2005

Molecular characterization of del(8)(p23.1p23.1) in a case of congenital diaphragmatic hernia.

A 36-week-old fetus was referred to the medical center because of his cystic mass and fluid in left thoracic cavity, and was delivered by cesarean section to manage neonatal problems at 37 weeks of gestation. Emergent surgical repair of the left diaphragmatic hernia was performed, but severe hypoxia persisted, and he expired on the following day. Chromosome analysis of cultured amniotic fluid cells indicated 46,XY,del(8)(p23.1p23.1). This is the fourth case of 8p23.1 deletion associated with diaphragmatic hernia. Microarray comparative genomic hybridization analysis using DNA of cultured amniotic fluid cells showed that six clones were deleted, which were mapped to the region between two low copy repeats (LCRs) at 8p23.1 previously described. Microsatellite analysis revealed that the deletion was of paternal origin, and his parents did not carry 8p23.1 polymorphic inversion. These data strongly suggested that the 8p23.1 interstitial deletion should have arisen through a different mechanism from that of inv dup del(8p) whose structural abnormality is always of maternal origin and accompanies heterozygous 8p23.1 polymorphic inversion in mother.

Adult↗
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↗