Unlike traditional techniques used to detect copy number gains and losses, which rely on the examination of a single target and prior knowledge of the region under investigation, CGH can be used to quickly scan an entire genome for imbalances. In addition, CGH does not require cells that are undergoing division (Speicher et al., 1993). However, as with earlier cytogenetic methods, the resolution of CGH has been limited to alterations of approximately 5-10 Mb for most clinical applications (Lichter et al., 2000; Kirchhoff et al., 1998).
Combining CGH with Microarrays: The Development of Array CGH
In an attempt to overcome some of the aforementioned limitations associated with traditional CGH, investigators have developed a newer method that combines the principles of CGH with the use of microarrays (Schena et al., 1995). Instead of using metaphase chromosomes, this method—which is known as array CGH, or simply aCGH—uses slides arrayed with small segments of DNA as the targets for analysis (Lucito et al., 2003). These microarrays are created by the deposit and immobilization of small amounts of DNA (known as probes) on a solid support, such as a glass slide, in an ordered fashion. Probes vary in size from oligonucleotides manufactured to represent areas of interest (25–85 base pairs) to genomic clones such as bacterial artificial chromosomes (80,000–200,000 base pairs). Because probes are several orders of magnitude smaller than metaphase chromosomes, the theoretical resolution of aCGH is proportionally higher than that of traditional CGH. The level of resolution is determined by considering both probe size and the genomic distance between DNA probes. For example, a microarray with probes selected from regions across the genome that are 1 Mb apart will be unable to detect copy number changes of the intervening sequence.
Regardless of the type of probe, the basic methodology for aCGH analysis is consistent (Figure 1). First, DNA is extracted from a test sample (e.g., blood, skin, fetal cells). The test DNA is then labeled with a fluorescent dye of a specific color, while DNA from a normal control (reference) sample is labeled with a dye of a different color. The two genomic DNAs, test and reference, are then mixed together and applied to a microarray. Because the DNAs have been denatured, they are single strands; thus, when applied to the slide, they attempt to hybridize with the arrayed single-strand probes. Next, digital imaging systems are used to capture and quantify the relative fluorescence intensities of the labeled DNA probes that have hybridized to each target. The fluorescence ratio of the test and reference hybridization signals is determined at different positions along the genome, and it provides information on the relative copy number of sequences in the test genome as compared to the normal genome. The recent sequencing of the human genome and development of high-throughput methods of robotically arraying genetic material on a solid surface have enabled the detection of submicroscopic chromosomal deletions and duplications at an unprecedented level (DeRisi et al., 1996; Schena et al., 1995; Shaffer et al., 2007).
Advantages of aCGH Technology
The primary advantage of aCGH is the ability to simultaneously detect aneuploidies, deletions, duplications, and/or amplifications of any locus represented on an array; in fact, one assay using this technique is equivalent to thousands of FISH experiments, with the attendant savings in labor and expense. In addition, aCGH has proven to be a powerful tool for the detection of submicroscopic chromosomal abnormalities in individuals with idiopathic mental retardation and various birth defects. Indeed, several large-scale studies demonstrate that aCGH has a 10%–20% detection rate of chromosomal abnormalities in children with mental retardation/developmental delay with or without congenital anomalies; only 3%–5% of these abnormalities would be detectable by other means. For example, in a study of 8,789 cases analyzed by aCGH, 1,049 (11.9%) had a clinically relevant chromosomal abnormality (Shaffer et al., 2007).
Studying Specific Chromosomal Regions with aCGH
Because aCGH facilitates simultaneous detection of multiple abnormalities and offers higher resolution than traditional cytogenetic methods, it has allowed investigators to focus on various types of rearrangements in particular regions of chromosomes. In recent years, aCGH has been particularly useful in the study of subtelomeric and pericentromeric rearrangements.
Studies of subtelomeric rearrangements illustrate how aCGH has revealed an unprecedented amount of information about the complexity of the human genome. Present on all but the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22, subtelomeric regions have been the subject of a great deal of study because they are relatively gene-rich (Saccone et al., 1992) and are prone to rearrangement by a number of mechanisms (Ballif et al., 2003, 2004). Moreover, rearrangement of subtelomeric regions has been suggested to represent a high proportion of abnormalities in individuals with idiopathic mental retardation.
The largest study of subtelomeric abnormalities to date examined 11,688 cases with subtelomeric FISH and detected pathogenic abnormalities in 2.6% (Ravnan et al., 2006). Interestingly, recent large-scale prospective studies using aCGH on similar populations show that interstitial deletions (which are caused by two breaks in the chromosome arm, the loss of the intervening segment, and the rejoining of the chromosome segments) are two to three times more frequent than terminal imbalances in subtelomeric regions (Shaw-Smith et al., 2007).
It is important to note that aCGH data can be verified using FISH analysis (Figure 2). For instance, Ballif and others (2007b) recently characterized 169 cases with subtelomeric abnormalities identified by aCGH. Although the coverage was sufficient to define the breakpoints in over half (56%) of the subtelomeric abnormalities, 44% of the abnormalities extended outside the coverage, suggesting that many such abnormalities are greater than 5 Mb in size. Of these 169 cases, 42 had interstitial deletions. These deletions would have been missed or incorrectly characterized by subtelomeric FISH panels that use a single clone to the most distal unique sequence for each region. In addition, six (3.5%) of the individuals had complex rearrangements that showed deletions along with duplications or additional deletions. The identification of these sorts of complex rearrangements suggests that chromosomal abnormalities are often more complex than previously thought.
The Future of aCGH
Array CGH has propelled cytogenetics from the microscope to the computer, combining CGH with high-throughput microarrays to simultaneously analyze hundreds or thousands of discrete regions of the genome and identify unbalanced karyotypes. Array CGH combines the locus-specific nature of FISH with the global genome view of high-resolution chromosomes; thus, this method represents the integration of traditional and molecular cytogenetic techniques and will continue to enable the clinical diagnosis of chromosomal abnormalities at an unprecedented resolution in the years to come.