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Apr 1, 2007 (Vol. 27, No. 7)

CGH for Detecting Copy Number Variation

High-Resolution Oligo Arrays for Research and BAC Arrays for Growing Diagnostics Sector

  • High density, oligonucleotide-based array-Comparative Genomic Hybridization (aCGH) technology is improving the capability to perform genome-wide DNA copy number assessment and to zoom in for a high-resolution look at targeted regions of the genome. These abilities are driving growth in the aCGH market and expanding the diversity of the customer base beyond the cytogenetics sector. The range of research applications includes basic biological studies, drug target discovery, and pharmacogenomics.

    Array CGH describes a technology in which microarrays comprised of well-defined DNA probes are used to screen DNA test samples to detect changes in sequence copy number. For whole-genome aCGH, genomic DNA isolated from a test and a reference sample are fluorescently labeled with two different colored dyes. It is allowed to hybridize to a microarray of whole-genome DNA attached to a polymer-coated glass slide. Regions of chromosomal DNA amplified or deleted in the test sample are identified based on the ratio of signals generated.

    In the research arena, interest in applying CGH “is growing by leaps and bounds,” says Varshal Dave, director of microarray and microdissection marketing at Molecular Devices (www.moleculardevices.com). The company’s GenePix® 4000B scanner with GenePix Pro analysis software scans microarrays at two wavelengths simultaneously using a dual laser system, simplifying the comparison of gene copy number between a reference and sample genome. Acquity® bioinformatics software provides a visual representation of the aCGH results by generating a drawing of all the chromosomes in the genome and color coding them according to calculated gene copy number ratios, including intron/exon coordinates if available.

    “People are interested in synthesizing data from copy number experiments with gene expression and microRNA data,” says Kevin Meldrum, marketing manager at Agilent Technologies (www.agilent.com). “It is all about developing a more comprehensive understanding of genomic aberrations and tracing their ultimate effect on gene regulation and transcription.”

    Oligo-array CGH also has a place in the clinical world, helping researchers reclassify and define genetic syndromes that remain undefined using conventional cytogenetics techniques, such as karyotyping or even BAC arrays.

    In the clinical diagnostics arena, oligo aCGH holds much promise for improving the ability to link poorly defined pediatric syndromes characterized by mental retardation and developmental delay with specific chromosomal copy number changes and rearrangements. A great deal of effort is also focused on understanding the role of copy number aberrations in coding and regulatory regions of the genome with predisposition to and occurrence of cancer. This technology will also open up new opportunities in prenatal and postnatal diagnostics, observes Meldrum. For now, though, bacterial artificial chromosome (BAC)-based CGH arrays are the standard for use in diagnostic applications.

    Meldrum points to three emerging needs in the CGH field: strategies for working with challenging samples, such as formalin-fixed paraffin-embedded (FFPE) tumor samples and cancer tissue samples that contain mixed (cancerous and normal) cell populations; advances in data analysis and visualization software that will make it possible to store, analyze, compare, and draw conclusions from large population studies; and the addition of capabilities to assess natural copy number variations (CNVs) in the context of disease research.

    Regarding information breakdown and visualization, Agilent is taking steps with the Human Genome CGH Microarray and accompanying analytics software. It provides genome-wide coverage across 244,000 60-mer probes with a spatial resolution averaging 2 kb, according to the company. The array technology can accommodate a starting genomic DNA samples as small as 25 ng, and Agilent’s CGH Analytics software allows for simultaneous analysis, visualization, and comparison of multiple CGH microarray profiles.

  • Redefining "Normal"

    Meldrum describes the ability to incorporate CNVs in aCGH as the next wave. In the same way that there is normal variation at the level of individual base pairs, or SNPs, that does not cause phenotypic changes, the growing recognition that the baseline of normal CNV may be greater than originally proposed has sparked efforts to characterize the extent of CNVs. Agilent plans to add CNV information to its aCGH products through refined probe design and new bioinformatics tools that will define the location and relevance of CNVs.

    A collaborative study emanating from the Sanger Institute, for example, explored global variation in copy number in the human genome using both array-based CGH and SNP genotyping, describing it as “functionally significant” and identifying 1,447 copy number variable regions (CNVRs) covering 360 megabases, or 12% of the genome. Richard Redon, et.al., noted that “the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution.”

    “Array CGH is a powerful addition to the repertoire of clinical cytogenetic methodologies but must be interpreted within the context of traditional cytogenetics,” remarks Mansoor Mohammed, Ph.D., CEO and president of CombiMatrix Molecular Diagnostics (CMDX; www.cmdiagnostics.com).

    When the results of aCGH reveal an extra copy of a genomic locus or of an entire chromosome, for example, it is important to understand whether the extra copy is due to a tandem duplication or a third copy conjoined with another chromosome, explains Dr. Mohammed. These possibilities are associated with different risks of recurrence (in future pregnancies) and may point to different etiologies of the chromosomal aberration. In many cases, traditional cytogenetics, guided by the findings of aCGH, are needed to determine if one parent carries a particular balanced translocation that manifests as an imbalance in the chromosome complement of the child.

    Fluorescence in situ hybridization (FISH) is the gold standard used to verify the accuracy of aCGH. FISH relies on BACs and other large insert clones. While oligo-based aCGH offers the potential advantage of generating a higher-resolution analysis, it is not yet ready for use in a clinical setting because oligos are incompatible with standard FISH methods, Dr. Mohammed cautions.

    “When you get a ratio from an oligo array suggesting a genome copy number aberration, it is difficult to confirm such aberrations within a chromosomal context,” he says. Furthermore, he points out that the higher resolution of oligo aCGH and its potential to detect smaller CNVs throughout the genome could inundate diagnosticians with large amounts of uninterpretable data until the background work has been done to define the scope of normal CNV in various populations.

    A sampling of the scientific literature reveals a growing knowledge base supporting the belief that CNVs, whether representing normal variation or linked to disease predisposition, are more common than previously recognized. Additionally, the number of papers on the application of aCGH in studies of model organisms continues to expand.

    A report in Genome Research (2007;17(3):337-347) describes the use of aCGH to screen for novel induced deletions in C. elegans and to determine the amount of natural gene content variation between two strains of C. elegans, shown to be almost 2%. The technology enabled detection of both large (50 kb) multigene deletions and of small (1 kb) single-gene deletions in homozygous and heterozygous samples.

    Timothy Graubert, M.D., and colleagues from Washington University in St. Louis along with NimbleGen Systems (www.nimblegen.com) generated a high-resolution map of segmental DNA CNV in the mouse genome, comparing 21 inbred mouse strains using aCGH. They identified CNVs ranging in size from 21 to 2,002 kb, many associated with known polymorphic traits, and were able to use copy number information to predict the phenotype of previously uncharacterized mouse strains.

  • The Diagnostic Frontier

    In 2005, the molecular diagnostics market for cancer alone was $315 million, and that is expected to rise to $1.35 billion by 2010, according to TSG Partners Strategic Analysis.

    “Array CGH is poised to become a powerful tool for clinical diagnostics,” says Eis. Routine karyotyping is only able to detect chromosomal rearrangements in the 5–10 megabase range, and the size of BAC clones do not allow mapping of regions smaller than about 100,000 base pairs. “Researchers have essentially hit a wall in CGH resolution with a BAC-array approach,” says Eis. This has fueled the transition to oligo-based aCGH.

    Flanking duplications of DNA segments can predispose chromosomal regions to recurrent rearrangement. This could result in deletion, duplication, or inversion of involved sequences that may be linked to phenotypes spanning pathologies associated with mental retardation, developmental delay, and morphologic abnormalities.

    A group of researchers investigating 130 target genomic regions in individuals with mental retardation (using BAC array CGH) identified 16 pathogenic rearrangements, including microdeletions in one region shared by four individuals (Nature Genetics 13 Aug 2006).

    Oligonucleotide array-based CGH allowed the researchers to refine the breakpoints of the microdeletion and define six contiguous genes deleted in all four subjects. They linked the breakpoint regions to sites of copy number polymorphism in control samples, suggesting that they may represent inherently unstable regions of the genome.

    Oligo array-based CGH is also proving to be a valuable tool for detecting and characterizing copy number aberrations in tumor samples, overcoming the limited resolution of conventional cancer cytogenetics. Fine-mapping of amplifications and deletions in a set of tumor samples allows researchers to refine common regions of gain or loss, thus narrowing down the set of cancer-related genes for a given locus, including down to the exon level.

    In the area of oncogenomics and understanding the link between DNA copy number changes and cancer type and progression, “there is a real need to move toward a paired diagnostics/therapeutics approach for cancer,” says Peg Eis, director of the array CGH product line at NimbleGen Systems.

    NimbleGen plans to introduce a 2.1-million feature array during second quarter 2007. The company’s current array products contain 385,000 unique long oligo probes (50–80 base pairs in length) designed to offer increased detection sensitivity and specificity. Its Maskless Array Synthesis (MAS) technology, based on use of a digital micromirror device (DMD), enables production of a new array within a day of creating a design, according to Eis.

    The company will also soon launch multiplex array formats, in which each slide can be partitioned into subarrays for simultaneous processing of multiple samples. For example, a 385,000 feature array can be used to analyze four samples with about 70,000 probes per subarray.

  • Contract aCGH Services

    Signature Genomic Laboratories (www.signaturegenomics.com) performs aCGH for diagnostic testing for mental retardation and developmental disabilities. The SignatureChip® is a targeted array comprised of 1,887 BAC clones. Designed to avoid most known CNVs, the chip interrogates 622 targeted loci and evaluates more than 70 recognized microdeletion or microduplication syndromes, 41 unique subtelomeric regions, and 43 unique pericentromic regions.

    “The main advantage of aCGH is that in a single overnight experiment you can detect every imbalance—gains or losses of DNA—that you would find with G-banding, FISH, or other methods,” says Lisa Shaffer, Ph.D., cofounder and CEO of Signature Genomic. “Our yield is more than two times that of a typical cytogenetics lab.”

    Among children with global developmental delay, about 3% of cases will show a chromosomal abnormality on conventional cytogenetic testing, whereas Signature Genomic can detect abnormalities in about 7% of cases, according to Dr. Shaffer. While linking a disorder to a chromosomal abnormality is an important diagnostic accomplishment in itself, some causes of mental retardation or developmental delay may be linked with a subclinical medical disease or with a predisposition to cancer, which might otherwise go undetected.

    Signature Genomic is not currently applying aCGH to cancer diagnostics, but Dr. Shaffer describes the technology as having tremendous potential for oncogenomics. In cancer, the diagnostic power will come from combining knowledge of genomic changes and of gene expression changes, she says.

    In September 2006, CombiMatrix Molecular Diagnostics brought its first molecular diagnostics test to the U.S. market. The Constitutional Genetic Array Test (CGAT), based on a BAC-CGH platform, can identify more than 50 common genomic abnormalities associated with developmental disorders in children, according to the CombiMatrix.

    The company recently released its clinically validated hematological malignancy test. CombiMatrix also leverages its technology for the rapid production of customizable oligo arrays by selling its proprietary desktop machines for array production, producing arrays (CustomArray™) for research use on a contract basis, and generating oligo array detection systems to detect infectious agents in test samples for use by government agencies, such as the Department of Defense, and through its diagnostics division, the company is developing a portfolio of BAC CGH arrays for clinical applications.

    For now, BAC arrays represent the standard for clinical diagnostic applications. However, Dr. Mohammed anticipates that within three to five years, as studies using oligo arrays yield a dataset that can differentiate ostensibly benign genomic variants from those either associated with a disease state or predictive of disease predisposition, high-density oligo aCGH will begin to penetrate the large and lucrative diagnostics market.

    Moving forward, as researchers define the scope of global genomic CNVs, they will then want to zoom in on specific chromosomal regions that are hotspots for copy number abnormalities to measure more accurately the size of the DNA segments affected and to identify the breakpoints at which a duplication or deletion starts and stops. This, Dr. Mohammed predicts, will drive demand for technology capable of rapidly producing customized oligo arrays and for multiplex arrays to facilitate screening of large numbers of samples.

  • Expanding the Toolbox

    Affymetrix (www.affymetrix.com) has increased the content of its genotyping microarrays from 10K to the current 500K product over a three-year period and plans to launch its 1-million density array by the end of the second quarter 2007 for whole genome association studies. Marcus Hausch, Ph.D., academic marketing manager for DNA products, says, “We strongly believe you have to look at the whole genome to understand the whole picture, both copy number changes and LOH, or loss of heterozygosity.”

    Using the company’s GeneChip® Human Mapping set and GeneChip Chromosome Copy Number Analysis, to combine genotyping and copy number changes“you can detect copy neutral events such as uniparental disomy,” adds Dr. Hausch, and distinguish between different LOH mechanisms. Access to LOH information can enable genome-wide hierarchical LOH clustering, which has proven useful for classifying cancers.

    BioPrime® Total Genomic Labeling Systems from Invitrogen (www.invitrogen.com) are all-inclusive kits designed for labeling genomic DNA for aCGH (BAC or oligo-based) applications. Starting with input DNA of 50 ng to 3 µg, the kit yields about 8 µg of labeled DNA, according to Invitrogen.

    Noting increasing demand for its BioPrime DNA Labeling Kit, Invitrogen realized that many customers were using components of the kit to produce arrays for CGH experiments. The new BioPrime Total system combines the DNA labeling and purification components needed to prepare labeled DNA for hybridization to aCGH probes. Jason Johnson, product manager for DNA microarrays at Invitrogen, says the higher concentration of exo-Klenow, new dye linker chemistry, and Alexa Fluor® 3 and 5 dyes optimized for aCGH microarray applications are of particular advantage. “Better dye incorporation yields a better signal on arrays,” notes Johnson.

    Spectral Genomics (www.spectralgenomics.com) offers the 1-megabase Spectral Chip™ 2600, a whole genome array comprised of BAC clones, and the Constitutional Chip™, which contains 434 BAC clones that span chromosomal regions known to be hot spots for DNA sequence amplifications or deletions associated with specific disorders or syndromes characterized by mental retardation and developmental delay.

    The Life Sciences division of TeleChem International, called ArrayIt®(www.arrayit.com), provides products and services to the microarray industry (GEN, October 15, 2006, pg.1). The company’s H25K human genome microarray is derived from 25,509 human genes. ArrayIt leverages TeleChem’s Next Generation Screening™ (NGS) technology, in which DNA samples from individual patients are spotted at unique locations on a microarray, and the array is then exposed to the genomic probes.

    QArray is a family of microarraying instruments from Genetix (www.genetix.com) that includes the QArrayMini for studies of single chromosomes or defined regions of a genome and the QArray2 and QArraymax for whole genome analysis. The GenoSensor™ Microarray System from Vysis (www.vysis.com), a wholly owned subsidiary of Abbott Laboratories, performs whole genome assessment for CNV and includes a digital image-based reader and software.

    For clinical applications, Blue Gnome(www.cambridgebluegnome.com) offers CytoChips BAC microarrays, capable of both whole genome screening at 850-kb resolution and more focused analysis of regions corresponding to 90 genetic conditions at 100 kb resolution. The company’s BlueFuse software for microarray analysis applies statistical modeling to distinguish signal from noise.

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