Genome-wide Fashion Research Articles

Analysis of the progression of systolic blood pressure using imputation of missing phenotype values.

We present a genome-wide association study of a quantitative trait, "progression of systolic blood pressure in time," in which 142 unrelated individuals of the Genetic Analysis Workshop 18 real genotype data were analyzed. Information on systolic blood pressure and other phenotypic covariates was missing at certain time points for a considerable part of the sample. We observed that the dropout process causing missingness is not independent of the initial systolic blood pressure; that is, the data is not missing completely at random. However, after the adjustment for age, the impact of systolic blood pressure on dropouts was no longer significant. Therefore, we decided to impute missing phenotype values by using information from individuals with complete phenotypic data. Progression of systolic blood pressure (∆SBP/∆t) was defined based on the imputed phenotypes and analyzed in a genome-wide fashion. We also conducted an exhaustive genome-wide search for interaction between single-nucleotide polymorphisms (7.14 × 1010 tests) under an allelic model.The suggested data imputation and the association analysis strategy proved to be valid in the sense that there was no evidence of genome-wide inflation or increased type I error in general. Furthermore, we detected 2 single-nucleotide polymorphisms (SNPs) that met the criterion for genome-wide significance (p≤5 × 10−8), which was also confirmed via Monte-Carlo simulation. In view of the rather small sample size, however, the results have to be followed-up in larger studies.

Transcript mapping based on dRNA-seq data.

BackgroundRNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems.ResultsWe present RNAseg, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori.ConclusionsWith our algorithm it is possible to identify operons and 5’- and 3’-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics.

Genome-wide association tests of inversions with application to psoriasis

Although inversions have occasionally been found to be associated with disease susceptibility through interrupting a gene or its regulatory region, or by increasing the risk for deleterious secondary rearrangements, no association study has been specifically conducted for risks associated with inversions, mainly because existing approaches to detecting and genotyping inversions do not readily scale to a large number of samples. Based on our recently proposed approach to identifying and genotyping inversions using principal components analysis (PCA), we herein develop a method of detecting association between inversions and disease in a genome-wide fashion. Our method uses genotype data for single nucleotide polymorphisms (SNPs), and is thus cost-efficient and computationally fast. For an inversion polymorphism, local PCA around the inversion region is performed to infer the inversion genotypes of all samples. For many inversions, we found that some of the SNPs inside an inversion region are fixed in the two lineages of different orientations and thus can serve as surrogate markers. Our method can be applied to case-control and quantitative trait association studies to identify inversions that may interrupt a gene or the connection between a gene and its regulatory agents. Our method also offers a new venue to identify inversions that are responsible for disease-causing secondary rearrangements. We illustrated our proposed approach to case-control data for psoriasis and identified novel associations with a few inversion polymorphisms.

Extent of linkage disequilibrium in large-breed dogs: chromosomal and breed variation

The aim of this study was to better define the extent of linkage disequilibrium (LD) in populations of large-breed dogs and its variation by breed and chromosomal region. Understanding the extent of LD is a crucial component for successful utilization of genome-wide association studies and allows researchers to better define regions of interest and target candidate genes. Twenty-four Golden Retriever dogs, 28 Rottweiler dogs, and 24 Newfoundland dogs were genotyped for single-nucleotide polymorphism (SNP) data using a high-density SNP array. LD was calculated for all autosomes using Haploview. Decay of the squared correlation coefficient (r (2)) was plotted on a per-breed and per-chromosome basis as well as in a genome-wide fashion. The point of 50% decay of r (2) was used to estimate the difference in extent of LD between breeds. Extent of LD was significantly shorter for Newfoundland dogs based upon 50% decay of r (2) data at a mean of 344kb compared to Golden Retriever and Rottweiler dogs at 715 and 834kb, respectively (P<0.0001). Notable differences in LD by chromosome were present within each breed and not strictly related to the length of the corresponding chromosome. Extent of LD is breed and chromosome dependent. To our knowledge, this is the first report of SNP-based LD for Newfoundland dogs, the first report based on genome-wide SNPs for Rottweilers, and an almost tenfold improvement in marker density over previous genome-wide studies of LD in Golden Retrievers.

The Kinase-Independent, Second Life of CDK6 in Transcription

CDK6 is an oncogenic kinase regulating the cell cycle. In this issue of Cancer Cell, Kollmann and colleagues demonstrate that CDK6 performs a kinase-independent transcriptional function in regulating expression of VEGF-A and p16INK4a. These observations link the cell cycle machinery and angiogenesis and reveal the presence of a fail-safe antiproliferative mechanism.

Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV's latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

Differential genome-wide profiling of tandem 3′ UTRs among human breast cancer and normal cells by high-throughput sequencing

Tandem 3' UTRs produced by alternative polyadenylation (APA) play an important role in gene expression by impacting mRNA stability, translation, and translocation in cells. Several studies have investigated APA site switching in various physiological states; nevertheless, they only focused on either the genes with two known APA sites or several candidate genes. Here, we developed a strategy to study APA sites in a genome-wide fashion with second-generation sequencing technology which could not only identify new polyadenylation sites but also analyze the APA site switching of all genes, especially those with more than two APA sites. We used this strategy to explore the profiling of APA sites in two human breast cancer cell lines, MCF7 and MB231, and one cultured mammary epithelial cell line, MCF10A. More than half of the identified polyadenylation sites are not included in human poly(A) databases. While MCF7 showed shortening 3' UTRs, more genes in MB231 switched to distal poly(A) sites. Several gene ontology (GO) terms and pathways were enriched in the list of genes with switched APA sites, including cell cycle, apoptosis, and metabolism. These results suggest a more complex regulation of APA sites in cancer cells than previously thought. In short, our novel unbiased method can be a powerful approach to cost-effectively investigate the complex mechanism of 3' UTR switching in a genome-wide fashion among various physiological processes and diseases.

How to build your own nextgen sequencing facility: the perspective of the diagnostic laboratory

The implementation of Next Generation Sequencing in the diagnostic laboratory presents unique opportunities and problems. For the first time it appears likely that a single instrument will be able to detect and characterise in parallel the common forms of genetic mutation and do so in a genome-wide fashion. For a diagnostic laboratory the instrumentation costs are high and the costs of consumables per run are expensive; therefore, the applications of the instrument need to be carefully designed to maximise yields of meaningful data. Issues of significance for the laboratory include data handling, accreditation and certification of bioinformaticians, standardisation of the data production pipeline and the approach to reporting the large numbers of variants identified.

Application of short hairpin RNAs (shRNAs) to study gene function in mammalian systems

Over the past decade, RNA interference (RNAi) plays an important role in biology, especially for silencing gene expression. RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Methods of mediating the RNAi effect involve small interfering RNA (siRNA) and short hairpin RNA (shRNA). In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. This article reviews the current knowledge on the mechanism and applications of shRNA in mammalian and human cells. Keyword: shRNA, siRNA, RNAi, dicer, gene silencing

EpiChIP: gene-by-gene quantification of epigenetic modification levels

The combination of chromatin immunoprecipitation with next-generation sequencing technology (ChIP-seq) is a powerful and increasingly popular method for mapping protein–DNA interactions in a genome-wide fashion. The conventional way of analyzing this data is to identify sequencing peaks along the chromosomes that are significantly higher than the read background. For histone modifications and other epigenetic marks, it is often preferable to find a characteristic region of enrichment in sequencing reads relative to gene annotations. For instance, many histone modifications are typically enriched around transcription start sites. Calculating the optimal window that describes this enrichment allows one to quantify modification levels for each individual gene. Using data sets for the H3K9/14ac histone modification in Th cells and an accompanying IgG control, we present an analysis strategy that alternates between single gene and global data distribution levels and allows a clear distinction between experimental background and signal. Curve fitting permits false discovery rate-based classification of genes as modified versus unmodified. We have developed a software package called EpiChIP that carries out this type of analysis, including integration with and visualization of gene expression data.

Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy number variation

DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray- and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10. We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Genome-Wide Association Study of Lp-PLA2 Activity and Mass in the Framingham Heart Study

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA2 activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA2 activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA2 activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6×10−24); CELSR2/PSRC1 on chromosome 1 (p = 3×10−15); SCARB1 on chromosome 12 (p = 1×10−8) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4×10−8). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA2 mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA2 activity and mass.

Use of high throughput sequencing to observe genome dynamics at a single cell level

With the development of high throughput sequencing technology, it becomes possible to directly analyze mutation distribution in a genome-wide fashion, dissociating mutation rate measurements from the traditional underlying assumptions. Here, we sequenced several genomes of Escherichia coli from colonies obtained after chemical mutagenesis and observed a strikingly nonrandom distribution of the induced mutations. These include long stretches of exclusively G to A or C to T transitions along the genome and orders of magnitude intra- and intergenomic differences in mutation density. Whereas most of these observations can be explained by the known features of enzymatic processes, the others could reflect stochasticity in the molecular processes at the single-cell level. Our results demonstrate how analysis of the molecular records left in the genomes of the descendants of an individual mutagenized cell allows for genome-scale observations of fixation and segregation of mutations, as well as recombination events, in the single genome of their progenitor.

High-Resolution Genome Wide Copy Number Alteration (CNA) and Loss of Heterozigosity (LOH) Analysis in Chronic Myeloid Leukemia (CML) Shows That High and Intermediate Sokal Risk Pts (Pts) Have Multiple Losses Targeting Genes Involved in DNA Repair.

Abstract 3262Poster Board III-1CML pts display a certain degree of clinical heterogeneity that is documented by the varying levels of response to tyrosine kinase inhibitor therapy and is best reflected by the Sokal risk score. Clinical differences must be a sign of some biological heterogeneity the basis of which, however, are still poorly understood. Today many high-throughput assays are available that allow to unravel the complexity of cancer cells in a genome-wide fashion. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution (<1 kb) karyotyping of DNA samples from 73 newly diagnosed chronic phase CML pts. Median age was 55 years (range, 25–73 years); male to female ratio was 39/34; pts were almost equally distributed by Sokal risk score (low, n=23; intermediate, n= 23 high, n= 27). Of 189 genes known to be implicated in the cellular DNA repair pathways, 135 (71%) were found to map in regions affected by CNAs or copy-neutral LOH (uniparental disomy, UPD) in 44/73 (60%) pts. However, this was markedly more frequent in high and intermediate Sokal risk pts (20/27, 74% and 16/23, 69%, respectively) than in low Sokal risk pts (8/23, 33%), although neither the total number of detected regions of CNAs/UPD per sample nor the QC parameters differ significantly across different risk categories. Regions of CNA involving DNA repair genes ranged from 105 Kb to 1.1 Mb and were either focal lesions involving a part or the whole single gene (17% of cases), or more extensive losses/gains including 2 to 84 genes. Monoallelic deletions were much more frequent than amplifications. Regions of UPD involving DNA repair genes were much larger and ranged from 980 kb to 32 Mb.The pathways and genes most frequently affected by CNAs or UPD are listed in the Table below:Base Excision Repair (BER)MUTYHDNA glycosilase1p34.1loss/upd10 ptsPNKPPolynucleotide kinase19q13.33loss10 ptsNEIL1DNA glycosilase15q24.2loss7 ptsPOLBDNA polymerase beta8p11.21loss/upd6 ptsPCNASliding clamp for DNA polymerases20p12.3loss6 ptsMismatch Repair (MMR)PMS2L5Mut L homolog - mismatch and loop recognition7q11.23loss/upd11 ptsMSH2Mut S homolog - mismatch and loop recognition2p21loss8 ptsPOLD1DNA polymerase delta19q13.33loss8 ptsPOLA2DNA polymerase alpha, subunit 22p16.3loss/upd6 ptsPOLE2DNA polymerase epsilon, subunit 214q21–22loss5 ptsNucleotide Excision Repair (NER)ERCC15' incision subunit of TFIIH complex19q13.32loss11 ptsERCC25' to 3'DNA helicase of TFIIH complex19q13.32loss10 ptsXAB2Transcription-coupled NER factor19p13.2loss/upd9 ptsCDK7Kinase subunit of TFIIH complex5q13.2loss7 ptsRPA4Binds damaged DNA in preincision complexesXp21.33loss/upd7 ptsRPA2Binds damaged DNA in preincision complexes12q24.31loss/upd6 ptsHomologous Recombination (HR)RAD51CHomologous pairing17q23.2loss/upd7 ptsRAD52Accessory factor for recombination12p13.33loss7 ptsXRCC2DNA break and crosslink repair7q36.1loss5 ptsNon-Homologous End Joining (NHEJ)PRKDCDNA-dependent protein kinase, catalytic subunit8q11.21loss/upd5 ptsDCLRE1CArtemis nuclease10p13loss5 ptsREV7DNA polymerase zeta, subunit1p36.22gain5 ptsOther genes involved in DNA replication/repair/modification or chromatin remodelingCHAF1AChromatin assembly factor19p13.3loss15 ptsRECQL5DNA helicase17q25.1loss/upd11 ptsRAD9BPCNA-like DNA damage sensor12q24.11loss/upd10 ptsCHEK2DNA-damage checkpoint22q12.1loss/upd8 ptsRAD17RFC-like DNA damage sensor5q13.2loss6 ptsTREX1DNAase III exonuclease3p12.31loss/upd6 ptsFor some genes (e.g., RAD52), the monoallelic deletion we detected was found to translate into reduced mRNA expression, observation that was also independently confirmed in an additional group of high/intermediate versus low Sokal risk pts. In all the 44 pts, multiple pathways and multiple genes within the same pathway were affected, supporting the hypothesis that the lesions we detected might actually have consequences on DNA integrity despite the known partial functional redundancy of pathways and effectors. For many of the genes identified in this screen, activating or inactivating mutations are known to occur, and together with overexpression or haploinsufficiency, have been linked to a mutator phenotype in several malignant conditions. We are currently investigating whether this may be the case also in CML. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures:Baccarani:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

Genome-wide profiling of genetic alterations in acute lymphoblastic leukemia: recent insights and future directions

Until recently, our understanding of the genetic factors contributing to the pathogenesis of acute lymphoblastic leukemia (ALL) has relied on the detection of gross chromosomal alterations and mutational analysis of individual genes. Although these approaches have identified many important abnormalities, they have been unable to identify the full repertoire of genetic alterations in ALL. The advent of high-resolution, microarray-based techniques to identify DNA copy number alterations and loss-of-heterozygosity in a genome-wide fashion has enabled the identification of multiple novel genetic alterations targeting key cellular pathways, including lymphoid differentiation, cell cycle, tumor suppression, apoptosis and drug responsiveness. Recent studies have extended these approaches to examine the biologic basis of high-risk ALL and treatment relapse. As these techniques continue to evolve and are integrated with genome-wide epigenetic and transcriptomic data, we will obtain a comprehensive understanding of the genetic and epigenetic alterations in ALL, and ultimately will be able to translate these findings into the development of novel therapeutic approaches directed against rational therapeutic targets. Here, we review recent data obtained from genome-wide profiling studies in ALL, and discuss potential avenues for future investigation.

Deletion of IKZF1 (Ikaros) Predicts Poor Outcome and Impaired Maturation in B-Progenitor Acute Lymphoblastic Leukemia

In genome-wide analyses of DNA copy number abnormalities (CNAs) and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, we previously reported a high frequency of genetic alterations of regulators of B lymphoid development and cell cycle in B-progenitor acute lymphoblastic leukemia (B-ALL), and a near-obligate deletion of the transcription factor Ikaros (IKZF1) in BCR-ABL1 ALL (Nature 2007; 446:758 and Nature 2008; 453:110). To assess the prognostic significance of genetic alterations and their effects on the leukemic cell transcriptome, we have now extended these studies to a cohort of 221 NCI high risk pediatric ALL cases treated on the Children's Oncology Group (COG) P9906 trial. The majority of these cases (N=170) lacked known cytogenetic abnormalities, and none were BCR-ABL1 positive. CNA and LOH data were generated using Affymetrix 500K SNP arrays, and gene expression data was available for 207 cases. Genomic resequencing was performed for PAX5 and IKZF1. These analyses revealed mutations in genes regulating B lymphoid development in 57.5% of cases, including PAX5 CNAs (31.7%), PAX5 sequence mutations (8.1%), IKZF1 deletions (24.9%) and IKZF1 sequence mutations (2.6%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation and drug response including CDKN2A/B (45.7%), RB1 (11.3%), BTG1 (10.4%), IL3RA (6.8%), KRAS (6.3%), NRAS (2%), NR3C1 (2%), NR3C2 (3%), and ERG (6.3%). To examine associations between genetic alterations and outcome in a genome-wide fashion, we used a semi-supervised principal components approach and identified a predictor of outcome driven by deletion/mutation of IKZF1, EBF, and BTLA (hazard ratio 3.74, P=8.1×10−5). IKZF1 lesions were most strongly associated with poor outcome (cumulative incidence of relapse 68.8% in IKZF1-mutated ALL vs. 29.1% for IKZF1 wild type cases, P=0.002). This copy number predictor also predicted outcome in an independent cohort of 258 B-progenitor ALL cases treated at St Jude Children's Research Hospital (HR 3.76, P=0.00023). Importantly, the St Jude cohort had a much higher proportion of cases with conventional recurring cytogenetic abnormalities including hyperdiploidy and translocations than the COG P9906 cohort (60.4% versus 23%, respectively), including BCR-ABL1 (N=21; 8.1%). Notably, the association of IKZF1 mutation and poor outcome was also observed in the cases lacking BCR-ABL1. The presence of an IKZF1 mutation was also associated with a high-level of minimal residual disease following induction therapy in both cohorts. We next defined a gene expression profile of high risk (IKZF1-mutated) ALL in each cohort. Using gene set enrichment analysis (GSEA), we found the high risk gene expression signature to be positively enriched for hematopoietic stem cell genes and genes associated with minimal residual disease, and negatively enriched for genes regulating cell cycle progression and genes upregulated during B cell maturation. Moreover, GSEA also demonstrated significant similarity of the expression signatures of high risk COG and St Jude ALL, a striking finding given the marked differences in sample composition of the two groups. As IKZF1 mutation is a near obligate lesion in BCR-ABL1 lymphoid leukemia (Nature 2008; 453:110) and in this study is a strong independent predictor of poor outcome in BCR-ABL1 negative ALL, we next examined similarity of the gene expression signatures of high risk (IKZF1-mutated) BCR-ABL1 negative ALL and BCR-ABL1 positive ALL. Using GSEA and direct comparison of differentially expressed genes, there was highly significant similarity between the two signatures. These findings indicate that IKZF1 mutation is a key determinant of the gene expression signature of both BCR-ABL1 positive and negative ALL, and is thus likely to be central to the pathogenesis and poor outcome of both leukemia subtypes. Thus, IKZF1 deletion is a new prognostic factor in ALL, and is associated with a gene expression profile indicative of impaired maturation of the leukemic blasts, an observation compatible with the known role of IKZF1 in regulating early lymphoid development. These findings have important implications for risk stratification of ALL.

Construction and application of a zebrafish array comparative genomic hybridization platform

The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development.