Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Abstract

Abstract. Comparative genomic in situ hybridization (CGH)provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from aCorrespondence to: T. Cremer patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.

Comparison of genomic abnormalities between BRCAX and sporadic breast cancers studied by comparative genomic hybridization

“…CGH analysis was carried out as described previously with minor modifications. 15 Briefly, biotin-labeled tumour DNA and digoxigenin-labeled reference DNA (Roche Diagnostics, Mannheim, Germany) were precipitated in the presence of Cot-1 DNA (Invitrogen, Karlsruhe, Germany) and salmon sperm DNA (Sigma, Munich, Germany) and hybridized for 3 days to normal metaphase spreads of a healthy donor. Probe detection was carried out using FITC for biotin-and Cy3 for digoxigenin-labeled DNA probes, respectively.…”

Chromosomal changes during development and progression of prostate adenocarcinomas

“…CGH analysis was performed according to Kallioniemi et al (1992) and du Manoir et al (1993) with modifcations. 600 ng of biotin-16-dUTP labelled DNA and 600 ng of SpectrumRed direct-labelled normal male DNA were simultaneously hybridized with 25 µg unlabelled Cot-1 DNA (Life Technologies Inc, Grand Island, NY) to denatured lymphocyte metaphases for 3 days.…”

Phyllodes tumors of the breast segregate in two groups according to genetic criteria

“…No chromosomal aberrations specific to phyllodes tumors have been identified so far. Using comparative genomic hybridization (CGH), a technique that allows a genome-wide screening of chromosome imbalances in a single experiment, 15,16 recurrent gains and losses, þ 1q, À3p, þ 7q, À6q and À3q, were found in phyllodes tumors. 17 Other molecular studies showed that somatic or germinal mutations of TP53 were present in isolated cases of malignant phyllodes tumors.…”