Array Painting

Although aCGH can identify copy number changes associated with chromosome rearrangement, aberrations that involve no loss or gain of DNA (inversions, balanced translo-

Fig. 3. Flow karyotype of a t(11;12) lymphoblastoid cell line. The derivative chromosomes are easily distinguished from the unaffected homologs by a change in DNA content and basepair ratio. (Adapted from ref. 20.)

cations) remain undetected. We have developed a modification of aCGH, which allows balanced translocations and their breakpoints to be analyzed on the microarrays. The method, which we have called array painting, involves the separation of the derivative chromosomes from the rest of the genome using flow sorting (20). On the flow cytometer, chromosomes in suspension and stained with two DNA-binding dyes can be identified using a combination of DNA content and basepair ratio. Most balanced translocations involve an unequal exchange of DNA between the two derivatives such that the derivative chromosomes are different in size and/or basepair ratio from the normal homologs (see Fig. 3). In this way, the derivative chromosomes can be sorted as purified fractions away from each other and their normal homologs, differentially labeled and hybridized to an array (see Fig. 4). On the array, fluorescence will only be detected on clones corresponding to the sequences present in the sorted chromosomes. The fluorescence ratio for these clones will either be high or low depending on which derivative chromosome the sequence of the clone corresponds (see Fig. 3). Should a clone on the array span a breakpoint, sequences from both the derivatives will hybridize generating intermediate ratio values.

We have used array painting together with aCGH to study patients with apparently balanced translocations expecting to map rapidly the translocation breakpoints and discover genes which might be disrupted by the rearrangement. However, we have discovered an unexpected level of complexity in these patients with as many as 60% of cases showing deletion at or near the translocation breakpoint, the involvement of additional chromosomes in the rearrangement or microdeletion/microduplication on chromosomes not involved in the translocation (21). The power of this methodology is its ability to screen the whole genome (aCGH) and the entire length of the derivative chromosomes (array painting) for rearrangement. This contrasts with the use of fluorescence in situ hybridization (FISH) for analysis of translocation breakpoints, which owing to the time and effort involved in performing FISH studies have focused primarily on the breakpoints, thus, potentially missing rearrangements elsewhere in the genome.

Fig. 4. Principle of Array painting. The graph shows a detail of the ratio plot for chromosome 11 close to the rearrangement breakpoint. Low ratios indicate sequences present on the derivative 11 and high ratios on the derivative 12. The analysis showed that the rearrangement involved an inversion/deletion event followed by translocation (for details, see ref. 20). (Adapted from ref. 20.)

Fig. 4. Principle of Array painting. The graph shows a detail of the ratio plot for chromosome 11 close to the rearrangement breakpoint. Low ratios indicate sequences present on the derivative 11 and high ratios on the derivative 12. The analysis showed that the rearrangement involved an inversion/deletion event followed by translocation (for details, see ref. 20). (Adapted from ref. 20.)

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