Application of array-based comparative genomic hybridization to clinical diagnostics

Abstract

Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that was recently adopted in the clinical laboratory. This technology was first developed as a research tool for the investigation of genomic alterations in cancer. It allows for a high-resolution evaluation of DNA copy number alterations associated with chromosome abnormalities. Array CGH is based on the use of differentially labeled test and reference genomic DNA samples that are simultaneously hybridized to DNA targets arrayed on a glass slide or other solid platform. In this review, we examine the technology and its transformation from a research tool into a maturing diagnostic instrument. We also evaluate the various approaches that have shaped the current platforms that are used for clinical applications. Finally, we discuss the advantages and shortcomings of "whole-genome" arrays and compare their diagnostic use to "targeted" arrays. Depending on their design, microarrays provide distinct advantages over conventional cytogenetic analysis because they have the potential to detect the majority of microscopic and submicroscopic chromosomal abnormalities. This new platform is poised to revolutionize modern cytogenetic diagnostics and to provide clinicians with a powerful tool to use in their increasingly sophisticated diagnostic capabilities.

Figure 1

Schematic representation of CGH microarray technology. Whole genomic DNA from a control or reference (left) and genomic DNA from a test or patient (right) are differentially labeled with two different fluorophores. The two genomic DNA samples are competitively cohybridized with large-insert clone DNA targets that have been robotically printed onto the microarray (middle). Computer imaging programs assess the relative fluorescence levels of each DNA for each target on the array (lower left). The ratio between control and test DNA for each clone can be linearly plotted using data analysis software to visualize dosage variations (lower right), indicated by a deviation from the normal log₂ ratio of zero.

Figure 2

Array CGH for chromosome 10 in three subjects. For each panel representing chromosome 10, each clone on the plot is arranged along the x axis according to its location on the chromosome with the most distal, telomeric short arm clones on the left and the most distal, telomeric long-arm clones on the right. The dark blue line represents the control/patient fluorescence intensity ratios for each clone, whereas the pink line represents the fluorescence intensity ratios obtained from a second hybridization in which the dyes have been reversed (patient/control). For a deletion, the blue line deviates up, and the pink line deviates down. For duplication (not shown), the pink line would deviate up, and the blue line would deviate down. For deletion, the deviation from zero is a ratio of 1:2 (patient/control). A: The plot for a normal chromosome 10. Note that all log₂ ratios for both experiments are zero. B: Terminal deletion of a single BAC clone at 10q26.3 (arrow). This deletion was demonstrated in a child and his clinically normal father, demonstrating a population/familial variant. C: Terminal deletion of six BAC clones at 10q26.3 (arrow). This deletion was de novo in origin and demonstrates the usefulness of multiple, contiguous clones from a genetic locus in providing clinical confidence in the laboratory results. In both cases (B and C), FISH confirmed the deletions. The single BAC deletion in (B) was familial and most likely of no clinical significance because it was inherited from a phenotypically normal parent. This illustrates our experience with most cases in which alteration of a single BAC clone at the telomere is often found in one of the parents and is therefore likely not to be clinically significant.