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Microarray-based Comparative Genomic Hybridization (array-CGH) has been applied for a decade to screen for submicroscopic DNA gains and losses in tumor and constitutional DNA samples. This method has become increasingly flexible with the integration of new biological resources generated by genome sequencing projects. In this chapter, we describe alternative strategies for whole genome screening and high resolution breakpoint mapping of copy number changes by array-CGH, as well as tools available for accurate analysis of array-CGH experiments. Although most methods listed here have been designed for microarrays comprising large-insert clones, they can be adapted easily to other types of microarray platforms, such as those constructed from printed or synthesized oligonucleotides.
Methods Mol Biol 2009
PMID:Comparative genomic hybridization: microarray design and data interpretation. 1938 71

Array-CGH involves the comparison of a test to a reference genome using a microarray composed of target sequences with known chromosomal coordinates. The test and reference DNA samples are used as templates to generate two probe DNAs labeled with distinct fluorescent dyes. The two probe DNAs are co-hybridized on a microarray in the presence of Cot-1 DNA to suppress unspecific hybridization of repeat sequences. After slide washes and drying, microarray images are acquired on a laser scanner and fluorescent intensities from every target sequence spot on the array are extracted using dedicated computer programs. Intensity ratios are calculated and normalized to enable data interpretation. Although the protocols explained in this chapter correspond primarily to the use of large-insert clone microarrays in either manual or automated fashion, necessary adaptations for hybridization on microarrays comprising shorter target DNA sequences are also briefly described.
Methods Mol Biol 2009
PMID:Comparative genomic hybridization: DNA labeling, hybridization and detection. 1938 74

The spatial resolution of microarray-based comparative genomic hybridization (array-CGH) is dependent on the length and density of target DNA sequences covering the chromosomal region of interest. Here we describe the methods developed at the Wellcome Trust Sanger Institute (Cambridge, UK) to construct microarrays comprising large-insert clones available through genome sequencing projects. These methods are applicable to Bacterial and Phage Artificial Chromosomes (BAC and PAC) as well as fosmid and cosmid clones. The protocols are scalable for the construction of microarrays composed of several hundreds up to several ten thousands clones.
Methods Mol Biol 2009
PMID:Comparative genomic hybridization: DNA preparation for microarray fabrication. 1938 75

Recent advances in DNA microarray technology have enabled researchers to comprehensively characterize the complex genomes of higher eukaryotic organisms at an unprecedented level of detail. Array-based comparative genomic hybridization (Array-CGH) has been widely used for detecting DNA copy number alterations on a genomic scale, where the mapping resolution is limited only by the number of probes on the DNA microarray. In this chapter, we present a validated protocol utilizing print-tip spotted HEEBO (Human Exonic Evidence Based Oligonucleotide) microarrays for conducting array-CGH using as little as 25 ng of genomic DNA from a wide variety of sources, including cultured cell lines and clinical specimens, with high spatial resolution and array-to-array reproducibility.
Methods Mol Biol 2009
PMID:Comparative genomic hybridization on spotted oligonucleotide microarrays. 1948 69

In this chapter, we introduce a few statistical algorithms for calling gains and losses in array-based comparative genomic hybridization (array CGH) data, including CBS, CLAC, CGHseg, and Fused Lasso. We illustrate the performance of the methods through simulated and real data examples. We also provide brief guidance on how to use the corresponding software at the end of this chapter.
Methods Mol Biol 2009
PMID:Algorithms for calling gains and losses in array CGH data. 1948 74

Central to systems biology are genome-wide technologies and high-throughput experimental approaches. Completion of the sequencing of the human genome as well as those of a number of other higher eukaryotes now allows for the first time the mapping of all of the cis-regulatory regions of genes as well as the details of nucleosome position and modification. One approach to achieving this goal involves chromatin immunoprecipitation combined with DNA oligonucleotide tiling arrays (ChIP-chip). This allows for the identification of genomic regions bound by a given factor, its cistrome, or harboring a given epigenomic modification through hybridization on tiling arrays covering the entire genome or specific regions of interest. This technology offers an unbiased assessment of the potential biological function of any DNA associated factor or epigenomic mark. Through integration of ChIP-chip data with complementary genome-wide approaches including expression profiling, CGH and SNP arrays, novel paradigms of transcriptional regulation and chromatin structure are emerging.
Methods Mol Biol 2009
PMID:Combining chromatin immunoprecipitation and oligonucleotide tiling arrays (ChIP-Chip) for functional genomic studies. 1948 77

Chromosomal aberrations resulting in aneuploidies have been implicated in the development of most cancers and numerous other genetic disorders. Aneuploidies are a key feature of genomic instability, so classification of these copy number changes will be important in understanding how rearrangements arise and how ongoing instability is maintained. Traditional methods for detecting copy number changes have relatively poor resolution, making accurate detection of breakpoints impossible. The advent of microarray technology and its advance over the years has improved the ability to detect aneuploidies with greater accuracy. Mammalian comparative genome hybridization on microarrays (array-CGH) has been applied to the study of many carcinomas, identifying common copy number changes in key regions including known oncogenes. However, the large size of mammalian genomes has made it impractical to perform whole genome CGH at high resolution. Yeast has been established as a useful model for studying pathways relevant to oncogenesis, particularly those that maintain the integrity of the genome. Given the smaller size of the yeast genome, oligonucleotide tiling arrays have been developed that allow for nucleotide resolution of the whole genome on a single chip. Here we describe in detail how to use these arrays to detect copy number variations in yeast. This method will be useful in many different studies, but particularly in monitoring and cataloguing the changes resulting from genetic instability.
Methods Mol Biol 2009
PMID:Comparative genome hybridization on tiling microarrays to detect aneuploidies in yeast. 1952 16

Uterine leiomyomas (ULs) are benign monoclonal tumors originating from myometrial tissue in the uterus. Genetic pathways that lead to myometrial transformation into leiomyomas are largely unknown. Approximately 40% of ULs are karyotypically abnormal by G-banding; however, the remaining 60% of leiomyomas do not contain cytogenetically visible genomic rearrangements. Recent technological advances such as array based comparative genomic hybridization (array CGH) and dense single nucleotide polymorphism (SNP) arrays have enabled genome-wide scanning for genomic rearrangements missed by karyotype banding analysis. In the current study, we employed a high resolution SNP microarray on 16 randomly selected ULs and normal myometrium samples to detect submicroscopic (<5 Mb) chromosomal aberrations. The SNP array identified gene dosage changes in 56% of the fibroids (9/16), 25% of which (4/16) had aberrations >5 Mb, whereas 31% of which (5/16) contained only submicroscopic copy number changes (<5 Mb). We corroborated 3/5 submicroscopic changes using quantitative PCR, meaning that ultimately, 19% of our samples (3/16) were found to contain only submicroscopic changes. Novel submicroscopic aberrations on chromosomal segments 1q42.13, 11q13.1 and 13q12.13 and large, previously unreported deletions on 15q11.2-q23, 17p-q21.31 and 22q12.2-q12.3 were identified. Previously reported deletions on 1p, 3q, 7q, 13, and chromosome 14q were also noted. RHOU, MAP3K11 and WASF3 gene copy numbers were changed in the subset of leiomyomas with submicroscopic aberrations, and these genes have previously been implicated in tumorigenesis. Our findings support the hypothesis that a significant fraction of ULs without visible cytogenetic changes harbor submicroscopic genomic rearrangements which may in turn contribute to transformation of normal myometrial tissue into leiomyomas.
Mol Hum Reprod 2009 Sep
PMID:Detection of novel copy number variants in uterine leiomyomas using high-resolution SNP arrays. 1956 54

The number of relevant and well-characterized cell lines and xenograft models for studying human breast cancer are few, and may represent a limitation for this field of research. With the aim of developing new breast cancer model systems for in vivo studies of hormone dependent and independent tumor growth, progression and invasion, and for in vivo experimental therapy studies, we collected primary mammary tumor specimens from patients, and implanted them in immunodeficient mice. Primary tumor tissue from 29 patients with breast cancer was implanted subcutaneously with matrigel in SCID mice, in the presence of continuous release of estradiol. The tumors were transferred into new animals when reaching a diameter of 15mm and engrafted tumors were harvested for morphological and molecular characterization from passage six. Further, gene expression profiling was performed using Agilent Human Whole Genome Oligo Microarrays, as well as DNA copy number analysis using Agilent Human Genome CGH 244K Microarrays. Of the 30 primary tumors implanted into mice (including two implants from the same patient), two gave rise to viable tumors beyond passage ten. One showed high expression levels of estrogen receptor-alpha protein (ER) while the other was negative. Histopathological evaluation of xenograft tumors was carried out at passage 10-12; both xenografts maintained the morphological characteristics of the original tumors (classified as invasive grade III ductal carcinomas). The genomic profile of the ER-positive xenograft tumor resembled the profile of the primary tumor, while the profile obtained from the ER-negative parental tumor was different from the xenograft. However, the ER-negative parental tumor and xenograft clustered on the same branch using unsupervised hierarchical clustering analysis on RNA microarray expression data of "intrinsic genes". A significant variation was observed in the expression of extracellular matrix (ECM)-related genes, which were found downregulated in the engrafted tumors compared to the primary tumor. By IHC and qRT-PCR we found that the downregulation of stroma-related genes was compensated by the overexpression of such molecules by the mouse host tissue. The two established breast cancer xenograft models showed different histopathological characteristics and profound diversity in gene expression patterns that in part can be associated to their ER status and here described as basal-like and luminal-like phenotype, respectively. These two new breast cancer xenografts represent useful preclinical tools for developing and testing of new therapies and improving our knowledge on breast cancer biology.
Mol Oncol 2009 Dec
PMID:Molecular profiling and characterization of luminal-like and basal-like in vivo breast cancer xenograft models. 1971 61

OTC deficiency, a partially dominant X-linked trait, is the most frequent inborn error of the urea cycle. We describe a female patient with a contiguous gene deletion syndrome encompassing the OTC, DMD, RPGR, CYBB and XK genes, amongst others, only manifesting features of OTC deficiency. Molecular characterization was ascertained by MLPA and confirmed by CGH microarray, which revealed an 8.7 Mb deletion of the X-chromosome. Complete de novo deletion of the OTC gene led to a severe clinical phenotype in the proband. The application of high resolution molecular genetic techniques such as MLPA and array CGH, in mutation negative OTC cases allows the identification of chromosomal rearrangements, such as large deletions and provides information for accurate genetic counseling and prenatal diagnosis.
Mol Genet Metab 2010 Jan
PMID:Contiguous gene deletion syndrome in a female with ornithine transcarbamylase deficiency. 1978 89


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