Differentially painting human chromosome arms with combined binary ratio-labeling fluorescence in situ hybridization

Abstract

Recently we developed a novel strategy for differentially painting all 24 human chromosomes. It is termed COBRA-FISH, short for combined binary ratio labeling-fluorescence in situ hybridization. COBRA-FISH is distinct from the pure combinatorial approach in that only 4 instead of 5 fluorophores are needed to achieve color discrimination of 24 targets. Furthermore, multiplicity can be increased to 48 by introduction of a fifth fluorophore. Here we show that color identification by COBRA-FISH of all of the p and q arms of human chromosomes is feasible, and we apply the technique for detecting and elucidating intra- and interchromosomal rearrangements. Compared with 24-color whole chromosome painting FISH, PQ-COBRA-FISH considerably enhances the ability to determine the composition of rearranged chromosomes as demonstrated by the identification of pericentric inversions and isochromosomes as well as the elucidation of the arm identity of chromosomal material involved in complex translocations that occur in solid tumors.

Figure 1

Chromosomal arms differentially painted by PQ-COBRA–FISH. PQ-COBRA–FISH to normal male metaphase chromosomes. (A) shows the superimposed, pseudocolor images of the three fluorophores used for ratio labeling the WCPs in a karyogram format. The first binary label (B) differentiates the two identically ratio-labeled WCP sets. The karyogram was generated automatically on the basis of chromosomal WCP–FISH ratios and the presence (odd numbered chromosomes plus Y) or absence (even numbered chromosomes plus X) of the first binary color. The second binary label, differentiating p and q arms, is displayed in C. All q arm paints were labeled with the second binary label, except for chromosomes 4 and 7, for which p arm paints were used. Note the disturbance of the COBRA–FISH signals on the X chromosome by the Y paint.

Figure 2

Intrachromosomal rearrangements detected by PQ-COBRA –FISH. (A) An inversion of chromosome 12 in a normal carrier. In this balanced rearrangement, the distal region of the long arm is positioned on the end of the short arm of the rearranged chromosome. The segment of the short arm transferred to the long arm of the inv(12) could not be detected by us, nor in the original study by conventional cytogenetics or FISH with distal markers (Speleman et al 1993). (B) A der(1), composed exclusively of 1q material in a cervical cancer cell line with a complex karyotype. For each normal and rearranged homolog, the sequence of display is ratio image, the q arm paint (second binary label) and the thresholded image of the q-arm paint.

Figure 3

PQ-COBRA–FISH of a complex cancer karyotype This near-tetraploid cancer cell shows a range of 2–6 copies of each chromosome and 10 structural rearrangements. (A,B,C) The ratio-, first- and second-binary images of PQ- COBRA–FISH of the nonrearranged chromosomes. In contrast to Fig. 1, the first binary label is on the even numbered and X chromosomes. (D) The rearranged chromosomes present in the same cell. The rearrangements include two isochromosomes, a deletion, the two products of a reciprocal translocation [t(2;3)], four unbalanced translocations involving two chromosomes, and one three-way translocation. The composition of the rearranged chromosomes is shown next to the chromosomes. The t(2;5) illustrates how the binary colors discriminate chromosome number and arm identity of chromosomes that carry the same ratio-label.