Supplementary Figures 1-3 from Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer

Supplementary Tables 1-6 from Comparative Analyses of Chromosome Alterations in Soft-Tissue Metastases within and across Patients with Castration-Resistant Prostate Cancer

Supplementary Figure 1 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Supplementary Table 2 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

Supplementary Table 1 from Genomic Alterations Indicate Tumor Origin and Varied Metastatic Potential of Disseminated Cells from Prostate Cancer Patients

Array comparative genomic hybridization (CGH) is an excellent tool to scan the genome for copy number variations (CNVs) when used conscientiously. This article is intended to provide an understanding of the basic principles of array CGH and the different options available to the user to design their array CGH experiments. Specifically, the six subsections discuss the different array platforms available, test and reference DNA preparation, reference DNA choice, the basics of hybridization, data processing, and our current understanding of CNVs in the human genome.

Background: Mammalian olfactory receptors (ORs) are subject to a remarkable but poorly understood regime of transcriptional regulation, whereby individual olfactory neurons each express only one allele of a single member of the large OR gene family.Results: We performed a rigorous search for enriched sequence motifs in the largest dataset of OR promoter regions analyzed to date. We combined measures of cross-species conservation with databases of known transcription factor binding sites and ab initio motif-finding algorithms. We found strong enrichment of binding sites for the O/E family of transcription factors and for homeodomain factors, both already known to be involved in the transcriptional control of ORs, but did not identify any novel enriched sequences. We also found that TATA-boxes are present in at least a subset of OR promoters.Conclusions: Our rigorous approach provides a template for the analysis of the regulation of large gene families and demonstrates some of the difficulties and pitfalls of such analyses. Although currently available bioinformatics methods cannot detect all transcriptional regulatory elements, our thorough analysis of OR promoters shows that in the case of this gene family, experimental approaches have probably already identified all the binding factors common to large fractions of OR promoters.

. clicking here colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others . here following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles (this information is current as of July 16, 2010 ): The following resources related to this article are available online at www.sciencemag.org http://www.sciencemag.org/cgi/content/full/291/5507/1298 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/291/5507/1298/DC1 can be found at: Supporting Online Material http://www.sciencemag.org/cgi/content/full/291/5507/1298#otherarticles , 8 of which can be accessed for free: cites 21 articles This article 63 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/291/5507/1298#otherarticles 10 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/genetics Genetics : subject collections This article appears in the following

With the sequencing of the male-specific region of the chimpanzee Y chromosome, it is now possible to make comparisons with the human Y sequence and to learn more about the recent evolution of the human Y chromosome. The two sequences differ markedly in structure and gene content, indicating rapid evolution during the past 6 million years. This finding is at odds with the common view that Y chromosomes are essentially static structures that evolve only very slowly by genetic loss. Rather, renovation and remodelling dominate the evolution of human and chimpanzee Y chromosomes. Possible reasons for this extraordinary divergence include genetic hitchhiking effects, species-specific mating behaviours and the Y chromosome's role in sperm production. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. The sequencing of the male-specific region of the Y chromosome (MSY) in the chimpanzee and comparison between the MSYs of the two species now reveals that they differ radically in sequence structure and gene content, indicating rapid evolution over the past 6 million years. The human Y chromosome began to evolve from an autosome hundreds of millions of years ago, acquiring a sex-determining function and undergoing a series of inversions that suppressed crossing over with the X chromosome1,2. Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. Prevailing theories hold that Y chromosomes evolve by gene loss, the pace of which slows over time, eventually leading to a paucity of genes, and stasis3,4. These theories have been buttressed by partial sequence data from newly emergent plant and animal Y chromosomes5,6,7,8, but they have not been tested in older, highly evolved Y chromosomes such as that of humans. Here we finished sequencing of the male-specific region of the Y chromosome (MSY) in our closest living relative, the chimpanzee, achieving levels of accuracy and completion previously reached for the human MSY. By comparing the MSYs of the two species we show that they differ radically in sequence structure and gene content, indicating rapid evolution during the past 6 million years. The chimpanzee MSY contains twice as many massive palindromes as the human MSY, yet it has lost large fractions of the MSY protein-coding genes and gene families present in the last common ancestor. We suggest that the extraordinary divergence of the chimpanzee and human MSYs was driven by four synergistic factors: the prominent role of the MSY in sperm production, ‘genetic hitchhiking’ effects in the absence of meiotic crossing over, frequent ectopic recombination within the MSY, and species differences in mating behaviour. Although genetic decay may be the principal dynamic in the evolution of newly emergent Y chromosomes, wholesale renovation is the paramount theme in the continuing evolution of chimpanzee, human and perhaps other older MSYs.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Prostate cancers (PCa) exhibit a wide range of molecular and biological profiles. In concert with this heterogeneity, there is a wide spectrum of responses to therapy. One of the significant limitations in unraveling the complexities of PCa and designing/evaluating novel therapeutic strategies has been the lack of pre-clinical models that closely replicate the diversity seen in man. To overcome this limitation we have developed 25 unique PCa xenograft lines (LuCaP series) where the initiating implants were derived from primary tumors, soft-tissue metastases and bone metastases. This presentation will provide details of a comprehensive molecular and biological characterization of these lines, including their responses to therapy. Methods: Tumors were obtained either from radical prostatectomies or from our rapid autopsy program, which is designed specifically for the acquisition of metastatic foci. Tumor pieces were implanted into male, immune compromised mice. Characterization involves: (a) quantification of serum PSA levels, (b) basic histology, (c) tumor doubling time, (d) a panel of >15 biomarkers assessed by immunohistochemistry, (e) gene expression array profiles, (f) chromosomal losses and gains, (g) expression of the TMPRSS2:ERG fusion gene, (h) presence of the androgen receptor (AR) and its splice variants, (i) bone remodeling perturbations associated with tumor growth in bone, and (j) response to therapy, including androgen ablation and docetaxel. Results: Overall, 18% of the attempts to establish xenografts were successful resulting in 25 lines. As in the clinical scenario, these xenografts display a wide range of characterization profiles. While most are adenocarcinomas, 3 are neuroendocrine and all histologically resemble the originating clinical specimen. PSA serum levels can reach into the low thousands of ng/ml for a 1 g tumor. AR gene and protein expression is highly variable and >50% of the lines express a constitutively active AR splice variant. The gene expression profiles show multiple clustering patterns that include a close association of the xenograft with the clinical specimen, pairing of androgen dependent lines with their castrate-resistant offspring and a distinction between adenocarcinoma and neuroendocrine histologies. Five of the lines are PTEN negative and 9 express the TMPRSS2:ERG gene fusion. The bone remodeling response ranges from osteoblastic to lytic. Responses to androgen ablation and docetaxel range from long duration to no response. Conclusions: These 25 LuCaP PCa xenograft lines provide a highly diverse, yet clinically relevant, panel for the study of PCa biology. This diversity most importantly suggests that misleading conclusions can be drawn from use of only one or two PCa models. This is extremely important in the evaluation of new therapeutic strategies, especially those that target specific pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3242.

We report an evolutionary analysis of the V1R gene family across 37 mammalian genomes. V1Rs comprise one of three chemosensory receptor families expressed in the vomeronasal organ, and contribute to pheromone detection. We first demonstrate that Trace Archive data can be used effectively to determine V1R family sizes and to obtain sequences of most V1R family members. Analyses of V1R sequences from trace data and genome assemblies show that species-specific expansions previously observed in only eight species were prevalent throughout mammalian evolution, resulting in "semi-private" V1R repertoires for most mammals. The largest families are found in mouse and platypus, whose V1R repertoires have been published previously, followed by mouse lemur and rabbit (approximately 215 and approximately 160 intact V1Rs, respectively). In contrast, two bat species and dolphin possess no functional V1Rs, only pseudogenes, and suffered inactivating mutations in the vomeronasal signal transduction gene Trpc2. We show that primate V1R decline happened prior to acquisition of trichromatic vision, earlier during evolution than was previously thought. We also show that it is extremely unlikely that decline of the dog V1R repertoire occurred in response to selective pressures imposed by humans during domestication. Functional repertoire sizes in each species correlate roughly with anatomical observations of vomeronasal organ size and quality; however, no single ecological correlate explains the very diverse fates of this gene family in different mammalian genomes. V1Rs provide one of the most extreme examples observed to date of massive gene duplication in some genomes, with loss of all functional genes in other species.

Subtelomeres are concentrations of interchromosomal segmental duplications capped by telomeric repeats at the ends of chromosomes. The nature of the segments shared by different sets of human subtelomeres reflects their high rate of recent interchromosomal exchange. Here, we characterize the rearrangements incurred by the 15q subtelomere after it arose from a chromosome fission event in the common ancestor of great apes. We used FISH, sequencing of genomic clones, and PCR to map the breakpoint of this fission and track the fate of flanking sequence in human, chimpanzee, gorilla, orangutan, and macaque genomes. The ancestral locus, a cluster of olfactory receptor (OR) genes, lies internally on macaque chromosome 7. Sequence originating from this fission site is split between the terminus of 15q and the pericentromere of 14q in the great apes. Numerous structural rearrangements, including interstitial deletions and transfers of material to or from other subtelomeres, occurred subsequent to the fission, such that each species has a unique 15q structure and unique collection of ORs derived from the fission locus. The most striking rearrangement involved transfer of at least 200 kb from the fission-site region to the end of chromosome 4q, where much still resides in chimpanzee and gorilla, but not in human. This gross structural difference places the subtelomeric defect underlying facioscapulohumeral muscular dystrophy (FSHD) much closer to the telomere in human 4q than in the hybrid 4q-15q subtelomere of chimpanzee.

Abstract Androgen deprivation is the mainstay of therapy for progressive prostate cancer. Despite initial and dramatic tumor inhibition, most men eventually fail therapy and die of metastatic castration-resistant (CR) disease. Here, we characterize the profound degree of genomic alteration found in CR tumors using array comparative genomic hybridization (array CGH), gene expression arrays, and fluorescence in situ hybridization (FISH). Bycluster analysis, we show that the similarity of the genomic profiles from primary and metastatic tumors is driven by the patient. Using data adjusted for this similarity, we identify numerous high-frequency alterations in the CR tumors, such as 8p loss and chromosome 7 and 8q gain. By integrating array CGH and expression array data, we reveal genes whose correlated values suggest they are relevant to prostate cancer biology. We find alterations that are significantly associated with the metastases of specific organ sites, and others with CR tumors versus the tumors of patients with localized prostate cancer not treated with androgen deprivation. Within the high-frequency sites of loss in CR metastases, we find an overrepresentation of genes involved in cellular lipid metabolism, including PTEN. Finally, using FISH, we verify the presence of a gene fusion between TMPRSS2 and ERG suggested by chromosome 21 deletions detected by array CGH. We find the fusion in 54% of our CR tumors, and 81% of the fusion-positive tumors contain cells with multiple copies of the fusion. Our investigation lays the foundation for a better understanding of and possible therapeutic targets for CR disease, the poorly responsive and final stage of prostate cancer. [Cancer Res 2009;69(19):7793–802]

BACKGROUND:DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. RESULTS:Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. CONCLUSION:The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

Abstract Disseminated epithelial cells can be isolated from the bone marrow of a far greater fraction of prostate-cancer patients than the fraction of patients who progress to metastatic disease. To provide a better understanding of these cells, we have characterized their genomic alterations. We first present an array comparative genomic hybridization method capable of detecting genomic changes in the small number of disseminated cells (10–20) that can typically be obtained from bone marrow aspirates of prostate-cancer patients. We show multiple regions of copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain, and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from 11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of disseminated cells from patients with organ-confined disease. However, we identify changes shared by these samples with their corresponding primary tumors and prostate-cancer alterations reported in the literature, evidence that these cells, like those in advanced disease, are disseminated tumor cells (DTC). We also show that DTCs from patients with advanced and localized disease share several abnormalities, including losses containing cell-adhesion genes and alterations reported to associate with progressive disease. These shared alterations might confer the capability to disseminate or establish secondary disease. Overall, the spectrum of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and for variation in that capacity in DTCs from localized disease. Our analysis lays the foundation for elucidation of the relationship between DTC genomic alterations and progressive prostate cancer. [Cancer Res 2008;68(14):5599–608]

BACKGROUND:The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes. RESULTS:In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor. CONCLUSION:These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of similar to 50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.